Emma Kivuva

Methylation analysis of 79 patients with growth restriction reveals novel patterns of methylation change at imprinted loci

This study was an investigation of 79 patients referred to the Wessex Regional Genetics Laboratory with suspected Russell–Silver Syndrome or unexplained short stature/intra uterine growth restriction, warranting genetic investigation. Methylation status was analysed at target sequences within eleven imprinted loci (PLAGL1, IGF2R, PEG10, MEST1, GRB10, KCNQ1OT1, H19, IGF2P0, DLK1, PEG3, NESPAS). Thirty seven percent (37%) (29 of 79) of samples were shown to have a methylation abnormality. The commonest finding was a loss of methylation at H19 (23 of 29), as previously reported in Russell–Silver Syndrome. In addition, four of these patients had methylation anomalies at other loci, of whom two showed hypomethylation of multiple imprinted loci, and two showed a complete gain of methylation at IGF2R. This latter finding was also present in five other patients who did not have demonstrable changes at H19. In total, 7 of 79 patients showed a gain of methylation at IGF2R and this was significantly different from a normal control population of 267 individuals (P=0.002). This study in patients with growth restriction shows the importance of widening the epigenetic investigation to include multiple imprinted loci and highlights potential involvement of the IGF2R locus.

Mutations in NLRP5 are associated with reproductive wastage and multilocus imprinting disorders in humans

Human-imprinting disorders are congenital disorders of growth, development and metabolism, associated with disturbance of parent of origin-specific DNA methylation at imprinted loci across the genome. Some imprinting disorders have higher than expected prevalence of monozygotic twinning, of assisted reproductive technology among parents, and of disturbance of multiple imprinted loci, for which few causative trans-acting mutations have been found. Here we report mutations in NLRP5 in five mothers of individuals affected by multilocus imprinting disturbance. Maternal-effect mutations of other human NLRP genes, NLRP7 and NLRP2, cause familial biparental hydatidiform mole and multilocus imprinting disturbance, respectively. Offspring of mothers with NLRP5 mutations have heterogenous clinical and epigenetic features, but cases include a discordant monozygotic twin pair, individuals with idiopathic developmental delay and autism, and families affected by infertility and reproductive wastage. NLRP5 mutations suggest connections between maternal reproductive fitness, early zygotic development and genomic imprinting.

CHRNG genotype–phenotype correlations in the multiple pterygium syndromes

Background Germline mutations in the CHRNG gene that encodes the γ subunit of the embryonal acetylcholine receptor may cause the non-lethal Escobar variant (EVMPS) or the lethal form (LMPS) of multiple pterygium syndrome (MPS). In addition CHRNG mutations and mutations in other components of the embryonal acetylcholine receptor may present with fetal akinesia deformation sequence (FADS) without pterygia. Methods In order to elucidate further the role of CHRNG mutations in MPS/FADS, this study evaluated the results of CHRNG mutation analysis in 100 families with a clinical diagnosis of MPS/FADS. Results CHRNG mutations were identified in 11/41 (27%) of families with EVMPS and 5/59 (8%) with LMPS/FADS. Most patients with a detectable CHRNG mutation (21 of 24 (87.5%)) had pterygia but no CHRNG mutations were detected in the presence of central nervous system anomalies. Discussion The mutation spectrum was similar in EVMPS and LMPS/FADS kindreds and EVMPS and LMPS phenotypes were observed in different families with the same CHRNG mutation. Despite this intrafamilial variability, it is estimated that there is a 95% chance that a subsequent sibling will have the same MPS phenotype (EVMPS or LMPS) as the proband (though concordance is less for more distant relatives). Based on these findings, a molecular genetic diagnostic pathway for the investigation of MPS/FADS is proposed.

An exome sequencing strategy to diagnose lethal autosomal recessive disorders.

Rare disorders resulting in prenatal or neonatal death are genetically heterogeneous. For some conditions, affected fetuses can be diagnosed by ultrasound scan, but this is not usually possible until mid-gestation. There is often limited fetal DNA available for investigation. We investigated a strategy for diagnosing autosomal recessive lethal disorders in non-consanguineous pedigrees with multiple affected fetuses. Exome sequencing was performed to identify genes where each parent is heterozygous for a rare non-synonymous-coding or splicing variant. Putative pathogenic variants were tested for cosegregation in affected fetuses and unaffected siblings. In eight couples of European ancestry, we found on average 1.75 genes (range 0–4) where both parents were heterozygous for rare potentially deleterious variants. A proof-of-principle study detected heterozygous DYNC2H1 variants in a couple whose five fetuses had short-rib polydactyly. Prospective analysis of two couples with multiple pregnancy terminations for fetal akinesia syndrome was performed and a diagnosis was obtained in both the families. The first couple were each heterozygous for a previously reported GLE1 variant, p.Arg569His or p.Val617Met; both were inherited by their two affected fetuses. The second couple were each heterozygous for a novel RYR1 variant, c.14130-2A>G or p.Ser3074Phe; both were inherited by their three affected fetuses but not by their unaffected child. Biallelic GLE1 and RYR1 disease-causing variants have been described in other cases with fetal akinesia syndrome. We conclude that exome sequencing of parental samples can be an effective tool for diagnosing lethal recessive disorders in outbred couples. This permits early prenatal diagnosis in future pregnancies.

Microdeletion 9q22.3 syndrome includes metopic craniosynostosis, hydrocephalus, macrosomia, and developmental delay.

Basal cell nevus syndrome (BCNS), also known as Gorlin syndrome (OMIM #109400) is a well‐described rare autosomal dominant condition due to haploinsufficiency of PTCH1. With the availability of comparative genomic hybridization arrays, increasing numbers of individuals with microdeletions involving this locus are being identified. We present 10 previously unreported individuals with 9q22.3 deletions that include PTCH1. While 7 of the 10 patients (7 females, 3 males) did not meet strict clinical criteria for BCNS at the time of molecular diagnosis, almost all of the patients were too young to exhibit many of the diagnostic features. A number of the patients exhibited metopic craniosynostosis, severe obstructive hydrocephalus, and macrosomia, which are not typically observed in BCNS. All individuals older than a few months of age also had developmental delays and/or intellectual disability. Only facial features typical of BCNS, except in those with prominent midforeheads secondary to metopic craniosynostosis, were shared among the 10 patients. The deletions in these individuals ranged from 352 kb to 20.5 Mb in size, the largest spanning 9q21.33 through 9q31.2. There was significant overlap of the deleted segments among most of the patients. The smallest common regions shared among the deletions were identified in order to localize putative candidate genes that are potentially responsible for each of the non‐BCNS features. These were a 929 kb region for metopic craniosynostosis, a 1.08 Mb region for obstructive hydrocephalus, and a 1.84 Mb region for macrosomia. Additional studies are needed to further characterize the candidate genes within these regions.

Inherited dup(17)(p11.2p11.2): Expanding the phenotype of the Potocki–Lupski syndrome

Potocki–Lupski syndrome (PTLS, OMIM: 610883) is a microduplication syndrome characterized by infantile hypotonia, failure to thrive, cardiovascular malformations, developmental delay, intellectual disability, and behavior abnormalities, the latter of which can include autism spectrum disorder. The majority of individuals with PTLS harbor a de novo microduplication of chromosome 17p11.2 reciprocal to the common recurrent 3.6 Mb microdeletion in the Smith–Magenis syndrome critical region. Here, we report on the transmission of the PTLS duplication across two generations in two separate families. Individuals in these families presented initially with developmental delay, behavior problems, and intellectual disability. We provide a detailed review of the clinical and developmental phenotype of inherited PTLS in both families. This represents the second report (second and third families) of PTLS in a parent–child pair and exemplifies the under‐diagnosis of this and likely other genetic conditions in adults with intellectual disability and/or psychiatric disorders.

Diagnosis of lethal or prenatal-onset autosomal recessive disorders by parental exome sequencing.

Rare genetic disorders resulting in prenatal or neonatal death are genetically heterogeneous, but testing is often limited by the availability of fetal DNA, leaving couples without a potential prenatal test for future pregnancies. We describe our novel strategy of exome sequencing parental DNA samples to diagnose recessive monogenic disorders in an audit of the first 50 couples referred.

Clinical and genetic aspects of KBG syndrome

KBG syndrome is characterized by short stature, distinctive facial features, and developmental/cognitive delay and is caused by mutations in ANKRD11, one of the ankyrin repeat‐containing cofactors. We describe 32 KBG patients aged 2–47 years from 27 families ascertained via two pathways: targeted ANKRD11 sequencing (TS) in a group who had a clinical diagnosis of KBG and whole exome sequencing (ES) in a second group in whom the diagnosis was unknown. Speech delay and learning difficulties were almost universal and variable behavioral problems frequent. Macrodontia of permanent upper central incisors was seen in 85%. Other clinical features included short stature, conductive hearing loss, recurrent middle ear infection, palatal abnormalities, and feeding difficulties. We recognized a new feature of a wide anterior fontanelle with delayed closure in 22%. The subtle facial features of KBG syndrome were recognizable in half the patients. We identified 20 ANKRD11 mutations (18 novel: all truncating) confirmed by Sanger sequencing in 32 patients. Comparison of the two ascertainment groups demonstrated that facial/other typical features were more subtle in the ES group. There were no conclusive phenotype–genotype correlations. Our findings suggest that mutation of ANKRD11 is a common Mendelian cause of developmental delay. Affected patients may not show the characteristic KBG phenotype and the diagnosis is therefore easily missed. We propose updated diagnostic criteria/clinical recommendations for KBG syndrome and suggest that inclusion of ANKRD11 will increase the utility of gene panels designed to investigate developmental delay.

Diverse phenotypic consequences of mutations affecting the C-terminus of FLNA

Filamin A, the filamentous protein encoded by the X-linked gene FLNA, cross-links cytoskeletal actin into three-dimensional networks, facilitating its role as a signalling scaffold and a mechanosensor of extrinsic shear forces. Central to these functions is the ability of FLNA to form V-shaped homodimers through its C-terminal located filamin repeat 24. Additionally, many proteins that interact with FLNA have a binding site that includes the C-terminus of the protein. Here, a cohort of patients with mutations affecting this region of the protein is studied, with particular emphasis on the phenotype of male hemizygotes. Seven unrelated families are reported, with five exhibiting a typical female presentation of periventricular heterotopia (PH), a neuronal migration disorder typically caused by loss-of-function mutations in FLNA. One male presents with widespread PH consistent with previous male phenotypes attributable to hypomorphic mutations in FLNA. In stark contrast, two brothers are described with a mild PH presentation, due to a missense mutation (p.Gly2593Glu) inserting a large negatively charged amino acid into the hydrophobic dimerisation interface of FLNA. Co-immunoprecipitation, in vitro cross-linking studies and gel filtration chromatography all demonstrated that homodimerisation of isolated FLNA repeat 24 is abolished by this p.Gly2593Glu substitution but that extended FLNAGly2593Glu repeat 16–24 constructs exhibit dimerisation. These observations imply that other interactions apart from those mediated by the canonical repeat 24 dimerisation interface contribute to FLNA homodimerisation and that mutations affecting this region of the protein can have broad phenotypic effects.Key messages• Mutations in the X-linked gene FLNA cause a spectrum of syndromes.• Genotype-phenotype correlations are emerging but still remain unclear.• C-term mutations can confer male lethality, survival or connective tissue defects.• Mutations leading to the latter affect filamin dimerisation.• This deficit is compensated for by remotely acting domains elsewhere in FLNA.

Rapid prenatal diagnosis using targeted exome sequencing: a cohort study to assess feasibility and potential impact on prenatal counseling and pregnancy management

PurposeUnexpected fetal abnormalities occur in 2–5% of pregnancies. While traditional cytogenetic and microarray approaches achieve diagnosis in around 40% of cases, lack of diagnosis in others impedes parental counseling, informed decision making, and pregnancy management. Postnatally exome sequencing yields high diagnostic rates, but relies on careful phenotyping to interpret genotype results. Here we used a multidisciplinary approach to explore the utility of rapid fetal exome sequencing for prenatal diagnosis using skeletal dysplasias as an exemplar.MethodsParents in pregnancies undergoing invasive testing because of sonographic fetal abnormalities, where multidisciplinary review considered skeletal dysplasia a likely etiology, were consented for exome trio sequencing (both parents and fetus). Variant interpretation focused on a virtual panel of 240 genes known to cause skeletal dysplasias.ResultsDefinitive molecular diagnosis was made in 13/16 (81%) cases. In some cases, fetal ultrasound findings alone were of sufficient severity for parents to opt for termination. In others, molecular diagnosis informed accurate prediction of outcome, improved parental counseling, and enabled parents to terminate or continue the pregnancy with certainty.ConclusionTrio sequencing with expert multidisciplinary review for case selection and data interpretation yields timely, high diagnostic rates in fetuses presenting with unexpected skeletal abnormalities. This improves parental counseling and pregnancy management.