Louise Tee

Clinical and molecular genetic features of Beckwith–Wiedemann syndrome associated with assisted reproductive technologies

BACKGROUND Beckwith-Wiedemann syndrome (BWS) is a model imprinting disorder resulting from mutations or epigenetic events affecting imprinted genes at 11p15.5. Most BWS cases are sporadic and result from imprinting errors (epimutations) involving either of the two 11p15.5 imprinting control regions (IC1 and IC2). Previously, we and other reported an association between sporadic BWS and assisted reproductive technologies (ARTs). METHODS In this study, we compared the clinical phenotype and molecular features of ART (IVF and ICSI) and non-ART children with sporadic BWS. A total of 25 patients with post-ART BWS were ascertained (12 after IVF and 13 after ICSI). RESULTS Molecular genetic analysis revealed an IC2 epimutations (KvDMR1 loss of methylation) in 24 of the 25 children tested. Comparison of clinical features of children with post-ART BWS to those with non-ART BWS and IC2 defects revealed a lower frequency of exomphalos (43 versus 69%, P = 0.029) and a higher risk of neoplasia (two cases, P = 0.0014). As loss of methylation at imprinting control regions other than 11p15.5 might modify the phenotype of BWS patients with IC2 epimutations, we investigated differentially methylated regions (DMRs) at 6q24, 7q32 and 15q13 in post-ART and non-ART BWS IC2 cases (n = 55). Loss of maternal allele methylation at these DMRs occurred in 37.5% of ART and 6.4% of non-ART BWS IC2 defect cases. Thus, more generalized DMR hypomethylation is more frequent, but not exclusive to post-ART BWS. CONCLUSIONS These findings provide further evidence that ART may be associated with disturbed normal genomic imprinting in a subset of children.

Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome

Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator of the Rab3 pathway implicated in exocytic release of neurotransmitters and hormones, in 12 families with Micro syndrome. We hypothesize that the underlying pathogenesis of Micro syndrome is a failure of exocytic release of ocular and neurodevelopmental trophic factors.

Homozygous loss-of-function mutations in the gene encoding the dopamine transporter are associated with infantile parkinsonism-dystonia

Genetic variants of the SLC6A3 gene that encodes the human dopamine transporter (DAT) have been linked to a variety of neuropsychiatric disorders, particularly attention deficit hyperactivity disorder. In addition, the homozygous Slc6a3 knockout mouse displays a hyperactivity phenotype. Here, we analyzed 2 unrelated consanguineous families with infantile parkinsonism-dystonia (IPD) syndrome and identified homozygous missense SLC6A3 mutations (p.L368Q and p.P395L) in both families. Functional studies demonstrated that both mutations were loss-of-function mutations that severely reduced levels of mature (85-kDa) DAT while having a differential effect on the apparent binding affinity of dopamine. Thus, in humans, loss-of-function SLC6A3 mutations that impair DAT-mediated dopamine transport activity are associated with an early-onset complex movement disorder. Identification of the molecular basis of IPD suggests SLC6A3 as a candidate susceptibility gene for other movement disorders associated with parkinsonism and/or dystonic features.

Health and population effects of rare gene knockouts in adult humans with related parents

Rare gene knockouts in adult humans On average, most peoples genomes contain approximately 100 completely nonfunctional genes. These loss-of-function (LOF) mutations tend to be rare and/or occur only as a single copy within individuals. Narasimhan et al. investigated LOF in a Pakistani population with high levels of consanguinity. Examining LOF alleles that were identical by descent, they found, as expected, an absence of homozygote LOF for certain protein-coding genes. However, they also identified many homozygote LOF alleles with no apparent deleterious phenotype, including some that were expected to confer genetic disease. Indeed, one family had lost the recombination-associated gene PRDM9. Science, this issue p. 474 The total loss of protein-coding genes, even those with the potential to confer genetic diseases, can be tolerated. Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3222 British adults of Pakistani heritage with high parental relatedness, discovering 1111 rare-variant homozygous genotypes with predicted loss of function (knockouts) in 781 genes. We observed 13.7% fewer homozygous knockout genotypes than we expected, implying an average load of 1.6 recessive-lethal-equivalent loss-of-function (LOF) variants per adult. When genetic data were linked to the individuals’ lifelong health records, we observed no significant relationship between gene knockouts and clinical consultation or prescription rate. In this data set, we identified a healthy PRDM9-knockout mother and performed phased genome sequencing on her, her child, and control individuals. Our results show that meiotic recombination sites are localized away from PRDM9-dependent hotspots. Thus, natural LOF variants inform on essential genetic loci and demonstrate PRDM9 redundancy in humans.

Mutations in TTC37 Cause Trichohepatoenteric Syndrome (Phenotypic Diarrhea of Infancy)

BACKGROUND & AIMS Trichohepatoenteric syndrome (THES) is an autosomal-recessive disorder characterized by life-threatening diarrhea in infancy, immunodeficiency, liver disease, trichorrhexis nodosa, facial dysmorphism, hypopigmentation, and cardiac defects. We attempted to characterize the phenotype and elucidate the molecular basis of THES. METHODS Twelve patients with classic THES from 11 families had detailed phenotyping. Autozygosity mapping was undertaken in 8 patients from consanguineous families using 250,000 single nucleotide polymorphism arrays and linked regions evaluated using microsatellite markers. Linkage was confirmed to one region from which candidate genes were analyzed. The effect of mutations on protein production and/or localization in hepatocytes and intestinal epithelial cells from affected patients was characterized by immunohistochemistry. RESULTS Previously unrecognized platelet abnormalities (reduced platelet alpha-granules, unusual stimulated alpha granule content release, abnormal lipid inclusions, abnormal platelet canalicular system, and reduced number of microtubules) were identified. The THES locus was mapped to 5q14.3-5q21.2. Sequencing of candidate genes showed mutations in TTC37, which encodes the uncharacterized tetratricopeptide repeat protein, thespin. Bioinformatic analysis suggested thespin to be involved in protein-protein interactions or chaperone. Preliminary studies of enterocyte brush-border ion transporter proteins (sodium hydrogen exchanger 2, sodium hydrogen exchanger 3, aquaporin 7, sodium iodide symporter, and hydrogen potassium adenosine triphosphatase [ATPase]) showed reduced expression or mislocalization in all THES patients with different profiles for each. In contrast the basolateral localization of Na/K ATPase was not altered. CONCLUSIONS THES is caused by mutations in TTC37. TTC37 mutations have a multisystem effect, which may be owing to abnormal stability and/or intracellular localization of TTC37 target proteins.

Methylation analysis and diagnostics of Beckwith-Wiedemann syndrome in 1,000 subjects

BackgroundBeckwith-Wiedemann syndrome (BWS), a congenital overgrowth disorder with variable expressivity and a predisposition to tumorigenesis, results from disordered expression and/or function of imprinted genes at chromosome 11p15.5. There are no generally agreed clinical diagnostic criteria, with molecular studies commonly performed to confirm diagnosis. In particular, methylation status analysis at two 11p15.5 imprinting control centres (IC1 and IC2) detects up to 80% of BWS cases (though low-level mosaicism may not be detected). In order to evaluate the relationship between the clinical presentation of suspected BWS and IC1/2 methylation abnormalities we reviewed the results of >1,000 referrals for molecular diagnostic testing.ResultsOut of 1,091 referrals, 507 (46.5%) had a positive diagnostic test for BWS. The frequency of tumours was 3.4% in those with a molecular diagnosis of BWS. Previously reported genotype-phenotype associations with paternal uniparental disomy, IC1, and IC2 epimutation groups were confirmed and potential novel associations detected. Predictive values of previously described clinical diagnostic criteria were compared and, although there were differences in their sensitivity and specificity, receiver operating characteristic (ROC) analysis demonstrated that these were not optimal in predicting 11p15.5 methylation abnormalities. Using logistic regression, we identified clinical features with the best predictive value for a positive methylation abnormality. Furthermore, we developed a weighted scoring system (sensitivity 75.9%, and specificity 81.8%) to prioritise patients presenting with the most common features of BWS, and ROC analysis demonstrated superior performance (area under the curve 0.85, 95% CI 0.83 to 0.87) compared to previous criteria.ConclusionsWe suggest that this novel tool will facilitate selection of patients with suspected BWS for routine diagnostic testing and so improve the diagnosis of the disorder.

Epimutation profiling in Beckwith-Wiedemann syndrome: relationship with assisted reproductive technology

BackgroundBeckwith-Wiedemann syndrome (BWS) is a congenital overgrowth disorder associated with abnormalities in 11p15.5 imprinted genes. The most common cause is loss of methylation (epimutation) at the imprinting control centre 2 (IC2/KvDMR1). Most IC2 epimutations occur sporadically but an association with conception after assisted reproductive technologies (ART) has been reported. A subgroup of IC2 epimutation cases also harbour epimutations at other imprinting centres (ICs) outside of 11p15.5. We have investigated the relationship between these multiple epimutation cases (ME+), history of ART and clinical phenotype in a cohort of 187 BWS IC2 epimutation patients.ResultsMethylation analysis at PLAGL1, MEST and IGF2R ICs demonstrated an over-representation of patients with abnormally low methylation (8.5%, 12% and 6% respectively). At IGF2R some patients (2%) had gain of methylation but this was also detected in controls. Though there were no significant correlations between the methylation index (MIs) at the three ICs tested, a subset of patients appeared to be susceptible to multiple epimutations (ME+) and 21.2% of ME + patients had been conceived by ART compared to 4.5% (P = 0.0033) without additional epimutations. Methylation array profiling (Illumina Goldengate®) of patients and controls (excluding 11p15.5 loci) demonstrated significant differences between patients and controls. No significant associations were found between aspects of the BWS phenotype and individual epimutations but we describe a case presenting with a post-ART BWS-like phenotype in which molecular analysis demonstrated loss of paternal allele methylation at the 11p15.5 IC1 locus (IC1 regulates imprinting of IGF2 and H19). Loss of paternal allele methylation at the IC1 is the molecular finding associated with Silver-Russell syndrome whereas BWS is associated with gain of maternal allele methylation at IC1. Further analysis demonstrated epimutations at PLAGL1 and MEST consistent with the hypothesis that the presence of multiple epimutations may be of clinical relevance.ConclusionsThese findings suggest that the ME + subgroup of BWS patients are preferentially, but not exclusively, associated with a history of ART and that, though at present, there are no clear epigenotype-phenotype correlations for ME + BWS patients, non-11p15.5 IC epimutations can influence clinical phenotype.

Comparison of the clinical scoring systems in Silver–Russell syndrome and development of modified diagnostic criteria to guide molecular genetic testing

Background About half of all children with a clinical diagnosis of Silver–Russell syndrome (SRS) have a detectable molecular genetic abnormality (maternal uniparental disomy of chromosome upd(7)mat or hypomethylation of H19 differentially methylated region (DMR). The selection of children for molecular genetic testing can be difficult for non-specialists because of the broad phenotypic spectrum of SRS and the tendency of the facial features to mitigate during late childhood. Several clinical scoring systems for SRS have been developed by specialist researchers, but the utility of these for guiding molecular genetic testing in routine clinical practice has not been established. Objectives To evaluate the utility of four published clinical scoring systems for genetic testing in a cohort of patients referred to a clinical service laboratory. Patients Individuals with suspected SRS referred for molecular genetic testing of H19 DMR methylation status or upd(7)mat. Results 36 of 139 (25.9%) patients referred for testing had a genetic abnormality identified. Comparison of four published clinical scoring systems demonstrated that all included subjective criteria that could be difficult for the general clinician to assess. We developed a novel, simplified, scoring system utilising four objective, easily measured parameters that performed similarly to the most sensitive and specific published scoring system. Discussion Effective utilisation of genetic testing by clinicians without specialist clinical genetics training will be facilitated by the development of targeted testing protocols that are based on robust objective clinical features and are designed for use in a busy clinical practice rather than a research setting.

Promoter mutation is a common variant in GJC2-associated Pelizaeus-Merzbacher-like disease

Pelizaeus-Merzbacher-like disease (PMLD) is a clinically and genetically heterogeneous neurological disorder of cerebral hypomyelination. It is clinically characterised by early onset (usually infantile) nystagmus, impaired motor development, ataxia, choreoathetoid movements, dysarthria and progressive limb spasticity. We undertook autozygosity mapping studies in a large consanguineous family of Pakistani origin in which affected children had progressive lower limb spasticity and features of cerebral hypomyelination on MR brain imaging. SNP microarray and microsatellite marker analysis demonstrated linkage to chromosome 1q42.13-1q42.2. Direct sequencing of the gap junction protein gamma-2 gene, GJC2, identified a promoter region mutation (c.-167A>G) in the non-coding exon 1. The c.-167A>G promoter mutation was identified in a further 4 individuals from two families (who were also of Pakistani origin) with clinical and radiological features of PMLD in whom previous routine diagnostic screening of GJC2 had been reported as negative. A common haplotype was identified at the GJC2 locus in the three mutation-positive families, consistent with a common origin for the mutation and likely founder effect. This promoter mutation has only recently been reported in GJC2-PMLD but it has been postulated to affect the binding of the transcription factor SOX10 and appears to be a prevalent mutation, accounting for ~29% of reported patients with GJC2-PMLD. We propose that diagnostic screening of GJC2 should include sequence analysis of the non-coding exon 1, as well as the coding regions to avoid misdiagnosis or diagnostic delay in suspected PMLD.

Germline mutations in RYR1 are associated with foetal akinesia deformation sequence/lethal multiple pterygium syndrome

IntroductionFoetal akinesia deformation sequence syndrome (FADS) is a genetically heterogeneous disorder characterised by the combination of foetal akinesia and developmental defects which may include pterygia (joint webbing). Traditionally multiple pterygium syndrome (MPS) has been divided into two forms: prenatally lethal (LMPS) and non-lethal Escobar type (EVMPS) types. Interestingly, FADS, LMPS and EVMPS may be allelic e.g. each of these phenotypes may result from mutations in the foetal acetylcholine receptor gamma subunit gene (CHRNG). Many cases of FADS and MPS do not have a mutation in a known FADS/MPS gene and we undertook molecular genetic studies to identify novel causes of these phenotypes.ResultsAfter mapping a novel locus for FADS/LMPS to chromosome 19, we identified a homozygous null mutation in the RYR1 gene in a consanguineous kindred with recurrent LMPS pregnancies. Resequencing of RYR1 in a cohort of 66 unrelated probands with FADS/LMPS/EVMPS (36 with FADS/LMPS and 30 with EVMPS) revealed two additional homozygous mutations (in frame deletions). The overall frequency of RYR1 mutations in probands with FADS/LMPS was 8.3%.ConclusionsOur findings report, for the first time, a homozygous RYR1 null mutation and expand the range of RYR1-related phenotypes to include early lethal FADS/LMPS. We suggest that RYR1 mutation analysis should be performed in cases of severe FADS/LMPS even in the absence of specific histopathological indicators of RYR1-related disease.