Fetal hydrops and the Incremental yield of Next-generation sequencing over standard prenatal Diagnostic testing (FIND) study: prospective cohort study and meta-analysis.

OBJECTIVE: To determine the incremental yield of exome sequencing (ES) over chromosomal microarray analysis (CMA) or karyotyping in prenatally diagnosed non-immune hydrops fetalis (NIHF). METHODS: A prospective cohort study (comprising an extended group of the Prenatal Assessment of Genomes and Exomes (PAGE) study) was performed which included 28 cases of prenatally diagnosed NIHF undergoing trio ES following negative CMA or karyotyping. These cases were combined with data from a systematic review of the literature. MEDLINE, EMBASE, CINAHL and ClinicalTrials.gov databases were searched electronically (January 2000 to October 2020) for studies reporting on the incremental yield of ES over CMA or karyotyping in fetuses with prenatally detected NIHF. Inclusion criteria for the systematic review were: (i) at least two cases of NIHF undergoing sequencing; (ii) testing initiated based on prenatal ultrasound-based phenotype; and (iii) negative CMA or karyotyping result. The incremental diagnostic yield of ES was assessed in: (i) all cases of NIHF; (ii) isolated NIHF; (iii) NIHF associated with an additional fetal structural anomaly; and (iv) NIHF according to severity (i.e. two vs three or more cavities affected). RESULTS: In the extended PAGE study cohort, the additional diagnostic yield of ES over CMA or karyotyping was 25.0% (7/28) in all NIHF cases, 21.4% (3/14) in those with isolated NIHF and 28.6% (4/14) in those with non-isolated NIHF. In the meta-analysis, the pooled incremental yield based on 21 studies (306 cases) was 29% (95% CI, 24-34%; P < 0.00001; I2 = 0%) in all NIHF, 21% (95% CI, 13-30%; P < 0.00001; I2 = 0%) in isolated NIHF and 39% (95% CI, 30-49%; P < 0.00001; I2 = 1%) in NIHF associated with an additional fetal structural anomaly. In the latter group, congenital limb contractures were the most prevalent additional structural anomaly associated with a causative pathogenic variant, occurring in 17.3% (19/110) of cases. The incremental yield did not differ significantly according to hydrops severity. The most common genetic disorders identified were RASopathies, occurring in 30.3% (27/89) of cases with a causative pathogenic variant, most frequently due to a PTPN11 variant (44.4%; 12/27). The predominant inheritance pattern in causative pathogenic variants was autosomal dominant in monoallelic disease genes (57.3%; 51/89), with most being de novo (86.3%; 44/51). CONCLUSIONS: Use of prenatal next-generation sequencing in both isolated and non-isolated NIHF should be considered in the development of clinical pathways. Given the wide range of potential syndromic diagnoses and heterogeneity in the prenatal phenotype of NIHF, exome or whole-genome sequencing may prove to be a more appropriate testing approach than a targeted gene panel testing strategy. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

A report on the impact of rapid prenatal exome sequencing on the clinical management of 52 ongoing pregnancies: a retrospective review.

OBJECTIVE: Studies have shown that prenatal exome sequencing (PES) improves diagnostic yield in cases of fetal structural malformation. We have retrospectively analysed PES cases from two of the largest fetal medicine centres in the UK to determine the impact of results on management of a pregnancy. DESIGN: A retrospective review of clinical case notes. SETTING: Two tertiary fetal medicine centres. POPULATION: Pregnancies with fetal structural abnormalities referred to clinical genetics via a multidisciplinary team. METHODS: We retrospectively reviewed the notes of all patients who had undergone PES. DNA samples were obtained via chorionic villus sampling or amniocentesis. Variants were filtered using patient-specific panels and interpreted using American College of Medical Genetics guidelines. RESULTS: A molecular diagnosis was made in 42% (18/43) ongoing pregnancies; of this group, there was a significant management implication in 44% (8/18). A positive result contributed to the decision to terminate a pregnancy in 16% (7/43) of cases. A negative result had a significant impact on management in two cases by affirming the decision to continue pregnancy. CONCLUSIONS: We demonstrate that the results of PES can inform pregnancy management. Challenges include variant interpretation with limited phenotype information. These results emphasise the importance of the MDT and collecting phenotype and variant data. As this testing is soon to be widely available, we should look to move beyond diagnostic yield as a measure of the value of PES. TWEETABLE ABSTRACT: Prenatal exome sequencing can aid decision-making in pregnancy management; review ahead of routine implementation in NHS.

Non-immune fetal hydrops: etiology and outcome according to gestational age at diagnosis.

OBJECTIVE: Fetal hydrops is associated with increased perinatal morbidity and mortality. The etiology and outcome of fetal hydrops may differ according to the gestational age at diagnosis. The aim of this study was to evaluate the cause, evolution and outcome of non-immune fetal hydrops (NIFH), according to the gestational age at diagnosis. METHODS: This was a retrospective cohort study of all singleton pregnancies complicated by NIFH, at the Fetal Medicine Unit at St George's University Hospital, London, UK, between 2000 and 2018. All fetuses had detailed anomaly and cardiac ultrasound scans, karyotyping and infection screening. Prenatal diagnostic and therapeutic intervention, gestational age at diagnosis and delivery, as well as pregnancy outcome, were recorded. Regression analysis was used to test for potential association between possible risk factors and perinatal mortality. RESULTS: We included 273 fetuses with NIFH. The etiology of the condition varied significantly in the three trimesters. Excluding 30 women who declined invasive testing, the cause of NIFH was defined as unknown in 62 of the remaining 243 cases (25.5%). Chromosomal aneuploidy was the most common cause of NIFH in the first trimester. It continued to be a significant etiologic factor in the second trimester, along with congenital infection. In the third trimester, the most common etiology was cardiovascular abnormality. Among the 152 (55.7%) women continuing the pregnancy, 48 (31.6%) underwent fetal intervention, including the insertion of pleuroamniotic shunts, fetal blood transfusion and thoracentesis. Fetal intervention was associated significantly with lower perinatal mortality (odds ratio (OR), 0.30 (95% CI, 0.14-0.61); P < 0.001); this association remained significant after excluding cases with a diagnosis of anemia or infection (OR, 0.29 (95% CI, 0.13-0.66); P = 0.003). In 104 fetuses not undergoing active fetal intervention, the gestational age at diagnosis was the only parameter that was significantly associated with the risk of perinatal mortality (OR, 0.92 (95% CI, 0.85-0.99); P = 0.035), while the affected body cavity and polyhydramnios were not (P > 0.05). CONCLUSIONS: An earlier gestational age at diagnosis of NIFH was associated with an increased risk of aneuploidy and worse pregnancy outcome, including a higher risk of perinatal loss. Fetal therapy was associated significantly with lower perinatal mortality. © 2020 Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Counseling in fetal medicine: agenesis of the corpus callosum.

In this Review, we aim to provide up-to-date and evidence-based answers to common questions regarding the diagnosis and prognosis of prenatally detected agenesis of the corpus callosum (ACC). A systematic literature search was performed to identify all reports of ACC and reference lists of articles were identified. ACC involves partial or complete absence of the main commissural pathway that connects the two cerebral hemispheres, and can be isolated (with no other abnormalities) or complex (coexisting with other abnormalities). It is a rare finding and the prevalence is difficult to estimate because of selection bias in reported series. The corpus callosum (CC) can be assessed on ultrasound by direct visualization, but indirect features, such as ventriculomegaly, absence of the cavum septi pellucidi or widening of interhemispheric fissure, are often the reason for detection in a screening population. Careful imaging in a center with a high level of expertise is required to make a full assessment and to exclude coexisting abnormalities, which occur in about 46% of fetuses. When available, magnetic resonance imaging appears to be an important adjunct as it allows direct visualization. It can reduce false-positive rates on ultrasound and can confirm ACC, it can assess whether this is complete or partial and it can help in detecting coexisting brain abnormalities not seen on ultrasound. The overall rate of chromosomal abnormality in fetuses with ACC is 18%, but this high rate includes both isolated and complex ACC; more recent studies suggest that chromosomal abnormalities are rare in isolated cases. Nevertheless, postnatal follow-up studies suggest that about 15% of cases thought to be isolated prenatally were found to have associated abnormalities after birth. Neurodevelopmental outcome in isolated ACC was recently reported in a systematic review and suggested normal outcome in about 65-75% of cases. Findings need to be considered in light of the several limitations of existing studies, in terms of study design, selection bias, varying definitions and imaging protocols, ascertainment bias and lack of control groups. These uncertainties mean that antenatal counseling is difficult and further large prospective studies are needed.

Biallelic mutation of MSH2 in primary human cells is associated with sensitivity to irradiation and altered RAD51 foci kinetics.

BACKGROUND: Reports of differential mutagen sensitivity conferred by a defect in the mismatch repair (MMR) pathway are inconsistent in their conclusions. Previous studies have investigated cells established from immortalised human colorectal tumour lines or cells from animal models. METHODS: We examined primary human MSH2-deficient neonatal cells, bearing a biallelic truncating mutation in MSH2, for viability and chromosomal damage after exposure to DNA-damaging agents. RESULTS: MSH2-deficient cells exhibit no response to interstrand DNA cross-linking agents but do show reduced viability in response to irradiation. They also show increased chromosome damage and exhibit altered RAD51 foci kinetics after irradiation exposure, indicating defective homologous recombinational repair. DISCUSSION: The cellular features and sensitivity of MSH2-deficient primary human cells are broadly in agreement with observations of primary murine cells lacking the same gene. The data therefore support the view that the murine model recapitulates early features of MMR deficiency in humans, and implies that the variable data reported for MMR-deficient immortalised human cells may be due to further genetic or epigenetic lesions. We suggest caution in the use of radiotherapy for treatment of malignancies in individuals with functional loss of MSH2.

Posterior fossa malformation in fetuses: a report of 56 further cases and a review of the literature.

OBJECTIVE: The purposes of this study were to determine the outcome of fetuses diagnosed as having a posterior fossa abnormality (PFA) and to find out if there are associated features helpful in determining the prognosis. METHODS: This is a retrospective study of all posterior fossa abnormalities detected prenatally in our Units within the last 10 years. Fifty six patients were selected. Outcome data was collected from the Clinical Genetics Department records and the attending obstetrician or pediatrician. RESULTS: An enlarged cisterna magna (ECM, diameter greater than 10 mm at 18-23 gw) was detected in 22 fetuses, which was isolated in 14 cases. All the patients followed-up (n = 11) with isolated ECM were normal at birth (100%). Non-isolated ECM was present in 8 cases. Further information was available in 7, 5 (71%) of whom had a poor outcome. A Dandy Walker complex abnormality (DWC) was detected in 34 patients. The majority of them had a poor prognosis, 54% if isolated and 84% if non-isolated. CONCLUSIONS: Isolated ECM detected on prenatal scans has a favourable outcome, while DWC is associated with a very high chance of a poor prognosis.

Outcome of prenatally diagnosed agenesis of the corpus callosum.

OBJECTIVE: To investigate the natural history, associated abnormalities and outcome in fetuses diagnosed prenatally with agenesis of the corpus callosum (ACC). METHODS: A retrospective study of all cases of prenatally detected ACC was performed in patients referred to two tertiary units between January 1993 and October 2003. Associated abnormalities, pregnancy outcome and infant follow-up were recorded. RESULTS: ACC was diagnosed in 117 cases. In 82 (70%) cases this was associated with other fetal structural (n = 49) or chromosomal abnormalities (n = 33). ACC was classified as an isolated prenatal finding in 35 (30%) cases. Assuming normal development in all cases lost to follow-up, significant developmental delay was present in 36% (95% CI, 15-65%) of isolated ACC. Furthermore, developmental delay was present in all cases with ventriculomegaly of at least 15 mm and in one of four cases with ventricular measurements less than 15 mm. CONCLUSIONS: The outcome of prenatally detected ACC is mainly dependent on the presence or absence of associated anomalies. The full assessment of fetal ACC mandates karyotyping, MRI and a search for more subtle ultrasound features of certain genetic syndromes. In this series, at least 36% (95% CI, 15-65%) of cases with isolated ACC exhibited significant developmental delay when assessed postnatally.

Catel-Manzke syndrome: a case report of a female with severely malformed hands and feet. An extension of the phenotype or a new syndrome?

An unusual case of a female infant with Catel-Manzke syndrome is presented. Additional features not previously reported include three accessory ossicles at the bases or associated with the proximal phalanx of the index, middle, ring and little fingers bilaterally. There are also numerous bony abnormalities in both feet. Previous cases have shown no more than 2 accessory ossicles in the hand and these usually involve the index alone. The foot abnormalities are more extensive than any previously seen in this syndrome. This is only the 8th female case out of a total of 27 reported cases.

Late onset microcephaly: failure of prenatal diagnosis.

We present a case of recurrent primary developmental microcephaly of late onset, the prenatal diagnosis of which could not be achieved despite performing targeted serial ultrasound scans that revealed no obvious fetal abnormality. Serial scans for head measurements and detailed examination of the brain anatomy by both transabdominal and transvaginal sonography including color and power Doppler assessment revealed no obvious brain abnormality. Frontal lobe distance and thalamic frontal lobe distance at 36 weeks were on the 30th and 50th centiles, respectively. Growth velocity remained on the 50th centile up to 36 weeks; between 36 and 38 weeks measurements were between the 35th and 40th centiles. The infant was delivered by Cesarean section at 38 weeks on parental request. On examination after birth the head circumference was on the 9th centile, but the facies was that of a microcephalic child with a sloping forehead and neurologically he was severely abnormal. The adequacy of the normal reference ranges used is reviewed: the use of sex-specific growth charts at 38 weeks would have demonstrated the biparietal diameter and the head circumference to be on the 20th and 15th centiles, respectively, rather than just below the 40th centile. However, even sex-specific charts may not allow the recognition of a substantial number of affected fetuses.

Outcome of antenatally diagnosed talipes equinovarus in an unselected obstetric population.

OBJECTIVE: To investigate the natural history and outcome of fetal talipes diagnosed by routine ultrasound scanning at 18-23 weeks' gestation. PATIENTS AND METHODS: This was a retrospective study of 103 228 pregnancies undergoing routine ultrasound scanning at 18-23 weeks' gestation. A computer search was made to identify all cases of fetal talipes and the records of these patients were examined to determine the incidence of other defects and pregnancy outcome. RESULTS: The incidence of fetal talipes following routine ultrasound examination was 0.10% (107/103 228) and was bilateral in 64 (59.8%) and unilateral in 43 (40.2%) cases. In 52 (48.6%) cases, talipes was of complex etiology, as it was associated with other defects, while, in 55 (51.4%) cases, it was classified as idiopathic. In 19% of cases, an initial diagnosis of idiopathic talipes was changed to complex, because of the subsequent development of associated features. Perinatal death and long-term neurodevelopmental or musculoskeletal problems were significantly more common when the talipes was complex rather than idiopathic (odds ratio, 150; 95% confidence interval, 34-665). Adverse outcomes were also seen more frequently with bilateral compared to unilateral talipes (odds ratio, 3.44; 95% confidence interval, 1.50-7.90). CONCLUSION: The outcome of antenatally detected talipes is mainly dependent on the presence or absence of other defects. A significant proportion of cases, thought to be idiopathic at presentation, will develop associated complex features when reassessed on subsequent scans or postnatally.

The UK national study of magnetic resonance imaging as a method of screening for breast cancer (MARIBS).

The UK national study of magnetic resonance imaging as a method of screening for breast cancer (MARIBS) is in progress. The study design, accrual to date, and related research projects are described. Revised accrual rates and expected recruitment are given. 15 cancers have been detected to date, from a total of 1236 screening measurements. This event rate and the tumour grades reported are compared with recent reports from other studies in women at high risk of breast cancer.

Prenatal ultrasound diagnosis of Leroy I cell disease.

Leroy I cell disease is a rare autosomal recessive disorder which progressively leads to death within the first decade of life. Invasive prenatal diagnosis is possible but is only undertaken in families who have previously had an affected child. We describe the antenatal ultrasound diagnosis of the disease in a case referred at 30 weeks' gestation for suspected polyhydramnios.

Complete exon-intron structure of the RPGR-interacting protein (RPGRIP1) gene allows the identification of mutations underlying Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is a genetically heterogeneous autosomal recessive condition responsible for congenital blindness or greatly impaired vision since birth. So far, six LCA loci have been mapped but only 4 out of 6 genes have been identified. A genome-wide screen for homozygosity was conducted in seven consanguineous families unlinked to any of the six LCA loci. Evidence for homozygosity was found in two of these seven families at the 14q11 chromosomal region. Two retinal specific candidate genes were known to map to this region, namely the neural retina leucine zipper (NRL) and the retinitis pigmentosa GTPase regulator interacting protein (RPGRIP1). No mutation of the NRL gene was found in any of the two families. Thus, we determined the complete exon-intron structure of the RPGRIP1 gene. RPGRIP1 encompasses 24 coding exons, nine of which are first described here with their corresponding exon-intron boundaries. The screening of the gene in the two families consistent with linkage to chromosome 14q11 allowed the identification of a homozygous null mutation and a homozygous missense mutation, respectively. Further screening of LCA patients unlinked to any of the four already identified LCA genes (n=86) identified seven additional mutations in six of them. In total, eight distinct mutations (5 out of 8 truncating) in 8/93 patients were found. So far this gene accounts for eight out of 142 LCA cases in our series (5.6%).

Prenatal findings on ultrasound and X-ray in a case of overgrowth syndrome associated with increased nuchal translucency.

A case of prenatal diagnosis of an overgrowth syndrome at 30 weeks of gestation is reported. The diagnosis was suggested on the basis of increased fetal growth from 16 weeks onwards, advanced bone age, and characteristic facial features such as hypertelorism, broad forehead and small chin. The fetus presented at 12 weeks with a markedly increased nuchal translucency thickness and generalized skin edema, but normal karyotype. Serial ultrasound scans revealed brain abnormalities including mild unilateral ventriculomegaly and a cyst in the cavum septi pellucidi. The pregnancy was terminated at the parents' request at 32 weeks of gestation and postmortem examination confirmed the prenatal findings. This case demonstrates the possibility of prenatal diagnosis of early overgrowth syndromes and highlights the dilemma arising from the prenatal diagnosis of a non-lethal condition associated with an uncertain prognosis and poorly documented in utero.

Clinical studies on submicroscopic subtelomeric rearrangements: a checklist.

BACKGROUND: Submicroscopic subtelomeric chromosome defects have been found in 7.4% of children with moderate to severe mental retardation and in 0.5% of children with mild retardation. Effective clinical preselection is essential because of the technical complexities and cost of screening for subtelomere deletions. METHODS: We studied 29 patients with a known subtelomeric defect and assessed clinical variables concerning birth history, facial dysmorphism, congenital malformations, and family history. Controls were 110 children with mental retardation of unknown aetiology with normal G banded karyotype and no detectable submicroscopic subtelomeric abnormalities. RESULTS: Prenatal onset of growth retardation was found in 37% compared to 9% of the controls (p<0.0005). A higher percentage of positive family history for mental retardation was reported in the study group than the controls (50% v 21%, p=0.002). Miscarriage(s) were observed in only 8% of the mothers of subtelomeric cases compared to 30% of controls (p=0.028) which was, however, not significant after a Bonferroni correction. Common features (>30%) among subtelomeric deletion cases were microcephaly, short stature, hypertelorism, nasal and ear anomalies, hand anomalies, and cryptorchidism. Two or more facial dysmorphic features were observed in 83% of the subtelomere patients. None of these features was significantly different from the controls. Using the results, a five item checklist was developed which allowed exclusion from further testing in 20% of the mentally retarded children (95% CI 13-28%) in our study without missing any subtelomere cases. As our control group was selected for the "chromosomal phenotype", the specificity of the checklist is likely to be higher in an unselected group of mentally retarded subjects. CONCLUSIONS: Our results suggest that good indicators for subtelomeric defects are prenatal onset of growth retardation and a positive family history for mental retardation. These clinical criteria, in addition to features suggestive of a chromosomal phenotype, resulted in the development of a five item checklist which will improve the diagnostic pick up rate of subtelomeric defects among mentally retarded subjects.

Identification of mutations in the MSX2 homeobox gene in families affected with foramina parietalia permagna.

Foramina parietalia permagna (FPP) is an autosomal dominant condition characterized by cranial defects of the parietal bones. It can be present as an isolated feature, but it is also one of the characteristics of a contiguous gene syndrome associated with deletions on chromosome 11p11-p12. One of the proteins known to be involved in skull development is the MSX2 homeobox protein. Previously, MSX2 has been shown to be mutated in patients suffering from Boston type craniosynostosis. We have now analyzed the MSX2 gene in five families affected with FPP. An intragenic microsatellite marker did not reveal any recombination and a cumulated LOD score of +3.2 at theta = 0 was obtained. Sequence analysis further showed that in four out of five families an MSX2 mutation was responsible for the skull defect. Moreover, it appears that FPP is caused by haplo-insufficiency of the MSX2 gene. This implies that Boston type craniosynostosis and FPP are allelic variants of the same gene, with FPP caused by loss of MSX2 function and craniosynostosis Boston type due to gain of MSX2 function.

Mutation analysis and embryonic expression of the HLXB9 Currarino syndrome gene.

The HLXB9 homeobox gene was recently identified as a locus for autosomal dominant Currarino syndrome, also known as hereditary sacral agenesis (HSA). This gene specifies a 403-amino acid protein containing a homeodomain preceded by a very highly conserved 82-amino acid domain of unknown function; the remainder of the protein is not well conserved. Here we report an extensive mutation survey that has identified mutations in the HLXB9 gene in 20 of 21 patients tested with familial Currarino syndrome. Mutations were also detected in two of seven sporadic Currarino syndrome patients; the remainder could be explained by undetected mosaicism for an HLXB9 mutation or by genetic heterogeneity in the sporadic patients. Of the mutations identified in the 22 index patients, 19 were intragenic and included 11 mutations that could lead to the introduction of a premature termination codon. The other eight mutations were missense mutations that were significantly clustered in the homeodomain, resulting, in each patient, in nonconservative substitution of a highly conserved amino acid. All of the intragenic mutations were associated with comparable phenotypes. The only genotype-phenotype correlation appeared to be the occurrence of developmental delay in the case of three patients with microdeletions. HLXB9 expression was analyzed during early human development in a period spanning Carnegie stages 12-21. Signal was detected in the basal plate of the spinal cord and hindbrain and in the pharynx, esophagus, stomach, and pancreas. Significant spatial and temporal expression differences were evident when compared with expression of the mouse Hlxb9 gene, which may partly explain the significant human-mouse differences in mutant phenotype.