Expansion of the neurodevelopmental phenotype of individuals with EEF1A2 variants and genotype-phenotype study.

Translation elongation factor eEF1A2 constitutes the alpha subunit of the elongation factor-1 complex, responsible for the enzymatic binding of aminoacyl-tRNA to the ribosome. Since 2012, 21 pathogenic missense variants affecting EEF1A2 have been described in 42 individuals with a severe neurodevelopmental phenotype including epileptic encephalopathy and moderate to profound intellectual disability (ID), with neurological regression in some patients. Through international collaborative call, we collected 26 patients with EEF1A2 variants and compared them to the literature. Our cohort shows a significantly milder phenotype. 83% of the patients are walking (vs. 29% in the literature), and 84% of the patients have language skills (vs. 15%). Three of our patients do not have ID. Epilepsy is present in 63% (vs. 93%). Neurological examination shows a less severe phenotype with significantly less hypotonia (58% vs. 96%), and pyramidal signs (24% vs. 68%). Cognitive regression was noted in 4% (vs. 56% in the literature). Among individuals over 10 years, 56% disclosed neurocognitive regression, with a mean age of onset at 2 years. We describe 8 novel missense variants of EEF1A2. Modeling of the different amino-acid sites shows that the variants associated with a severe phenotype, and the majority of those associated with a moderate phenotype, cluster within the switch II region of the protein and thus may affect GTP exchange. In contrast, variants associated with milder phenotypes may impact secondary functions such as actin binding. We report the largest cohort of individuals with EEF1A2 variants thus far, allowing us to expand the phenotype spectrum and reveal genotype-phenotype correlations.

Faecal haemoglobin concentration in adenoma, before and after polypectomy, approaches the ideal tumour marker.

BACKGROUND: Polypectomy may be performed at colonoscopy and then subsequent surveillance undertaken. It is thought that faecal haemoglobin concentration (f-Hb), estimated by quantitative faecal immunochemical tests (FIT), might be a useful tumour marker. METHODS: Consecutive patients enrolled in colonoscopy surveillance were approached at two hospitals. A specimen for FIT was provided before colonoscopy and, ideally after 3 weeks, a second FIT sample from those who had polypectomy. A single FIT system (OC-Sensor io, Eiken Chemical Co., Ltd) was used to generate f-Hb. RESULTS: 1103 Patients were invited; 643 returned a FIT device (uptake: 58.3%). Four patients had known inflammatory bowel disease (IBD) and were excluded, leaving 639 (57.9%) with an age range of 25-90 years (median 64 years), 54.6% male. Of 593 patients who had a f-Hb result and completed colonoscopy, advanced neoplasia was found in 41 (6.9%); four colorectal cancer (CRC): 0.7% and 37 advanced adenoma (AA): 6.3%, and a further 127 (21.4%) had non-advanced adenoma (NAA). The median f-Hb was significantly greater in AA as compared to NAA; 6.0 versus 1.0 µg Hb/g faeces, p < 0.0001.134/164 (81.7%) of invited patients returned a second FIT device: 28 were patients with AA in whom median pre-polypectomy f-Hb was 19.2, falling to 3.5 µg Hb/g faeces post-polypectomy, p = 0.01, and 106 with NAA had median pre-polypectomy f-Hb 0.8 compared to 1.0 µg Hb/g faeces post-polypectomy, p = 0.96. CONCLUSIONS: Quantitative FIT could provide a good tumour marker in post-polypectomy surveillance, reduce colonoscopy requirements and minimise potential risk to patients.

Individuals with heterozygous variants in the Wnt-signalling pathway gene FZD5 delineate a phenotype characterized by isolated coloboma and variable expressivity.

BACKGROUND: Anophthalmia, microphthalmia and coloboma are a genetically heterogenous spectrum of developmental eye disorders. Recently, variants in the Wnt-pathway gene Frizzled Class Receptor 5 (FZD5) have been identified in individuals with coloboma and rarely microphthalmia, sometimes with additional phenotypes and variable penetrance. MATERIALS AND METHODS: We identified variants in FZD5 in individuals with developmental eye disorders from the UK (including the DDD Study [www.ddduk.org/access.html]), France and Spain using whole genome/exome sequencing or customized NGS panels of ocular development genes. RESULTS: We report eight new families with FZD5 variants and ocular coloboma. Three individuals presented with additional syndromic features, two explicable by additional variants in other genes (SLC12A2 and DDX3X). In two families initially showing incomplete penetrance, re-examination of apparently unaffected carrier individuals revealed subtle ocular colobomatous phenotypes. Finally, we report two families with microphthalmia in addition to coloboma, representing the second and third reported cases of this phenotype in conjunction with FZD5 variants. CONCLUSIONS: Our findings indicate FZD5 variants are typically associated with isolated ocular coloboma, occasionally microphthalmia, and that extraocular phenotypes are likely to be explained by other gene alterations.

Multiple Self-Healing Squamous Epithelioma (MSSE): A Digenic Trait Associated with Loss of Function Mutations in TGFBR1 and Variants at a Second Linked Locus on the Long Arm of Chromosome 9.

MSSE (Ferguson-Smith disease) is a rare familial condition in which multiple skin tumors resembling squamous carcinomas invade locally and then regress spontaneously after several months, leaving disfiguring scars. We review evidence from haplotype studies in MSSE families with common ancestry that the condition is caused by loss of function mutations in TGFBR1 interacting with permissive variants at a second linked locus on the long arm of chromosome 9. The spectrum of TGFBR1 mutations in MSSE and the allelic disorder Loeys Dietz syndrome (characterized by developmental anomalies and thoracic aortic aneurysms) differ. Reports of patients with both MSSE and Loeys Dietz syndrome are consistent with variants at a second locus determining whether self-healing epitheliomas occur in patients with the loss of function mutations found in most MSSE patients or the missense mutations in the intracellular kinase domain of TGFBR1 that characterize Loeys Dietz syndrome.

Non-Invasive Prenatal Diagnosis of Retinoblastoma Inheritance by Combined Targeted Sequencing Strategies.

Retinoblastoma, the most common childhood eye cancer, presents in two forms: heritable or sporadic. Heritable retinoblastoma is caused by a germline mutation in the RB1 gene. Early diagnosis of children at risk of inheriting an RB1 mutation is crucial to achieve optimal clinical outcome. Currently, the majority of genetic testing is performed on newborns, which has multiple disadvantages for both families and the healthcare system. We have developed a non-invasive prenatal diagnosis (NIPD) service for retinoblastoma, available from 8 weeks' gestation, which uses a combination of massively parallel sequencing (MPS) techniques, dependent on the inheritance model. Detection of paternal or suspected de novo RB1 variants is achieved through amplicon-based MPS. NIPD of a fetus at risk of maternal inheritance is performed using capture-based targeted sequencing and relative haplotype dosage analysis. In addition, we show proof of principle of how capture-based sequencing can be used for de novo variants unsuitable for amplicon-based testing. In total, we report the NIPD of 15 pregnancies, results of which show 100% concordance with all postnatal testing performed at the time of publication (n = 12) with remaining pregnancies ongoing. NIPD of retinoblastoma therefore offers a viable alternative to newborn genetic testing.

Correction: KAT6A Syndrome: genotype-phenotype correlation in 76 patients with pathogenic KAT6A variants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

SLC12A2 variants cause a neurodevelopmental disorder or cochleovestibular defect.

The SLC12 gene family consists of SLC12A1-SLC12A9, encoding electroneutral cation-coupled chloride co-transporters. SCL12A2 has been shown to play a role in corticogenesis and therefore represents a strong candidate neurodevelopmental disorder gene. Through trio exome sequencing we identified de novo mutations in SLC12A2 in six children with neurodevelopmental disorders. All had developmental delay or intellectual disability ranging from mild to severe. Two had sensorineural deafness. We also identified SLC12A2 variants in three individuals with non-syndromic bilateral sensorineural hearing loss and vestibular areflexia. The SLC12A2 de novo mutation rate was demonstrated to be significantly elevated in the deciphering developmental disorders cohort. All tested variants were shown to reduce co-transporter function in Xenopus laevis oocytes. Analysis of SLC12A2 expression in foetal brain at 16-18 weeks post-conception revealed high expression in radial glial cells, compatible with a role in neurogenesis. Gene co-expression analysis in cells robustly expressing SLC12A2 at 16-18 weeks post-conception identified a transcriptomic programme associated with active neurogenesis. We identify SLC12A2 de novo mutations as the cause of a novel neurodevelopmental disorder and bilateral non-syndromic sensorineural hearing loss and provide further data supporting a role for this gene in human neurodevelopment.

Faecal haemoglobin can define risk of colorectal neoplasia at surveillance colonoscopy in patients at increased risk of colorectal cancer.

BACKGROUND: Quantitative faecal immunochemical tests measure faecal haemoglobin concentration (f-Hb), which increases in the presence of colorectal neoplasia. OBJECTIVE: We examined the diagnostic accuracy of faecal immunochemical test (FIT)in patients at increased risk of colorectal cancer (CRC) attending for surveillance colonoscopy as per national guidelines. METHODS: A total of 1103 consecutive patients were prospectively invited to complete a FIT before their scheduled colonoscopy in two university hospitals in 2014- 2016. F-Hb was analysed on an OC-Sensor io automated analyser (Eiken Chemical Co., Ltd, Tokyo, Japan) with a limit of detection of 2 µg Hb/g faeces. The diagnostic accuracy of f-Hb for CRC and higher-risk adenoma was examined. RESULTS: A total of 643 patients returned a faecal test. After excluding 4 patients with known inflammatory bowel disease, 639 (57.9%) remained in the study: age range: 25-90 years (median: 64 years, interquartile range (IQR): 55-71): 54.6% male. Of 593 patients who also completed colonoscopy, 41 (6.9%) had advanced neoplasia (4 CRC, 37 higher-risk adenoma). Of the 238 patients (40.1%) who had detectable f-Hb, 31 (13.0%) had advanced neoplasia (2 CRC, 29 higher-risk adenoma) compared with 10 (2.8%) in those with undetectable f-Hb (2 CRC, 8 higher-risk adenoma). Detectable f-Hb gave negative predictive values of 99.4% for CRC and 97.2% for CRC plus higher-risk adenoma. CONCLUSION: In patients at increased risk of CRC under colonoscopy surveillance, a test measuring faecal haemoglobin can provide an objective estimate of the risk of advanced neoplasia, and could enable tailored scheduling of colonoscopy.

Opposite Modulation of RAC1 by Mutations in TRIO Is Associated with Distinct, Domain-Specific Neurodevelopmental Disorders.

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper- or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.

The phenotype of Sotos syndrome in adulthood: A review of 44 individuals.

Sotos syndrome is an overgrowth-intellectual disability (OGID) syndrome caused by NSD1 pathogenic variants and characterized by a distinctive facial appearance, an intellectual disability, tall stature and/or macrocephaly. Other associated clinical features include scoliosis, seizures, renal anomalies, and cardiac anomalies. However, many of the published Sotos syndrome clinical descriptions are based on studies of children; the phenotype in adults with Sotos syndrome is not yet well described. Given that it is now 17 years since disruption of NSD1 was shown to cause Sotos syndrome, many of the children first reported are now adults. It is therefore timely to investigate the phenotype of 44 adults with Sotos syndrome and NSD1 pathogenic variants. We have shown that adults with Sotos syndrome display a wide spectrum of intellectual ability with functioning ranging from fully independent to fully dependent. Reproductive rates are low. In our cohort, median height in adult women is +1.9 SD and men +0.5 SD. There is a distinctive facial appearance in adults with a tall, square, prominent chin. Reassuringly, adults with Sotos syndrome are generally healthy with few new medical issues; however, lymphedema, poor dentition, hearing loss, contractures and tremor have developed in a small number of individuals.

KAT6A Syndrome: genotype-phenotype correlation in 76 patients with pathogenic KAT6A variants.

PURPOSE: Pathogenic variants in KAT6A have recently been identified as a cause of syndromic developmental delay. Within 2 years, the number of patients identified with pathogenic KAT6A variants has rapidly expanded and the full extent and variability of the clinical phenotype has not been reported. METHODS: We obtained data for patients with KAT6A pathogenic variants through three sources: treating clinicians, an online family survey distributed through social media, and a literature review. RESULTS: We identified 52 unreported cases, bringing the total number of published cases to 76. Our results expand the genotypic spectrum of pathogenic variants to include missense and splicing mutations. We functionally validated a pathogenic splice-site variant and identified a likely hotspot location for de novo missense variants. The majority of clinical features in KAT6A syndrome have highly variable penetrance. For core features such as intellectual disability, speech delay, microcephaly, cardiac anomalies, and gastrointestinal complications, genotype- phenotype correlations show that late-truncating pathogenic variants (exons 16-17) are significantly more prevalent. We highlight novel associations, including an increased risk of gastrointestinal obstruction. CONCLUSION: Our data expand the genotypic and phenotypic spectrum for individuals with genetic pathogenic variants in KAT6A and we outline appropriate clinical management.

Features of multiple self-healing squamous epithelioma and Loeys-Dietz syndrome in a patient with a novel TGFBR1 variant.

Multiple self-healing squamous epithelioma (MSSE, Ferguson-Smith disease) and Loeys-Dietz syndrome (LDS) are allelic conditions associated with pathogenic variants in the transforming growth factor beta receptor 1 gene (TGFBR1). We describe a patient with a novel missense variant in this gene: c.664G > A, p.[Gly222Arg], who clinically presents with both syndromes. The patient also has a history of gastric antral vascular ectasia, which has not been reported previously in LDS.

Pathogenicity and Penetrance of Germline SDHA Variants in Pheochromocytoma and Paraganglioma (PPGL).

Germline SDHA mutations are reported in a minority of pheochromocytoma/paraganglioma (PPGL) cases but are associated with an increased risk of malignancy, leading some to advocate cascade genetic testing and surveillance screening of "at-risk" first-degree relatives. However, such approaches rely on accurate estimates of variant pathogenicity and disease penetrance, which may have been subject to ascertainment and reporting biases, although the recent provision of large population-based DNA sequence data sets may provide a potentially unbiased resource to aid variant interpretation. Thus, the aim of the current study was to evaluate the pathogenicity and penetrance of SDHA variants reported in literature-based PPGL cases by comparing their frequency to those occurring in the Genome Aggregation Database (GnomAD) data set, which provides high-quality DNA sequence data on 138,632 individuals. In total, 39 different missense or loss-of-function (LOF) SDHA variants were identified in 95 PPGL index cases. Notably, many of the PPGL-associated SDHA alleles were observed at an unexpectedly high frequency in the GnomAD cohort, with ~1% and ~0.1% of the background population harboring a rare missense or LOF variant, respectively. Although the pathogenicity of several SDHA alleles was supported by significant enrichment in PPGL cases relative to GnomAD controls, calculations of disease penetrance based on allele frequencies in the respective cohorts resulted in much lower estimates than previously reported, ranging from 0.1% to 4.9%. Thus, although this study provides support for the etiological role of SDHA in PPGL formation, it suggests that most variant carriers will not manifest PPGLs and are unlikely to benefit from periodic surveillance screening.

The Tatton-Brown-Rahman Syndrome: A clinical study of 55 individuals with de novo constitutive DNMT3A variants.

Tatton-Brown-Rahman syndrome (TBRS; OMIM 615879), also known as the DNMT3A-overgrowth syndrome, is an overgrowth intellectual disability syndrome first described in 2014 with a report of 13 individuals with constitutive heterozygous DNMT3A variants. Here we have undertaken a detailed clinical study of 55 individuals with de novoDNMT3A variants, including the 13 previously reported individuals. An intellectual disability and overgrowth were reported in >80% of individuals with TBRS and were designated major clinical associations. Additional frequent clinical associations (reported in 20-80% individuals) included an evolving facial appearance with low-set, heavy, horizontal eyebrows and prominent upper central incisors; joint hypermobility (74%); obesity (weight ³2SD, 67%); hypotonia (54%); behavioural/psychiatric issues (most frequently autistic spectrum disorder, 51%); kyphoscoliosis (33%) and afebrile seizures (22%). One individual was diagnosed with acute myeloid leukaemia in teenage years. Based upon the results from this study, we present our current management for individuals with TBRS.

Tumour risks and genotype-phenotype correlations associated with germline variants in succinate dehydrogenase subunit genes SDHB, SDHC and SDHD.

BACKGROUND: Germline pathogenic variants in SDHB/SDHC/SDHD are the most frequent causes of inherited phaeochromocytomas/paragangliomas. Insufficient information regarding penetrance and phenotypic variability hinders optimum management of mutation carriers. We estimate penetrance for symptomatic tumours and elucidate genotype-phenotype correlations in a large cohort of SDHB/SDHC/SDHD mutation carriers. METHODS: A retrospective survey of 1832 individuals referred for genetic testing due to a personal or family history of phaeochromocytoma/paraganglioma. 876 patients (401 previously reported) had a germline mutation in SDHB/SDHC/SDHD (n=673/43/160). Tumour risks were correlated with in silico structural prediction analyses. RESULTS: Tumour risks analysis provided novel penetrance estimates and genotype-phenotype correlations. In addition to tumour type susceptibility differences for individual genes, we confirmed that the SDHD:p.Pro81Leu mutation has a distinct phenotype and identified increased age-related tumour risks with highly destabilising SDHB missense mutations. By Kaplan-Meier analysis, the penetrance (cumulative risk of clinically apparent tumours) in SDHB and (paternally inherited) SDHD mutation-positive non-probands (n=371/67 with detailed clinical information) by age 60 years was 21.8% (95% CI 15.2% to 27.9%) and 43.2% (95% CI 25.4% to 56.7%), respectively. Risk of malignant disease at age 60 years in non-proband SDHB mutation carriers was 4.2%(95% CI 1.1% to 7.2%). With retrospective cohort analysis to adjust for ascertainment, cumulative tumour risks for SDHB mutation carriers at ages 60 years and 80 years were 23.9% (95% CI 20.9% to 27.4%) and 30.6% (95% CI 26.8% to 34.7%). CONCLUSIONS: Overall risks of clinically apparent tumours for SDHB mutation carriers are substantially lower than initially estimated and will improve counselling of affected families. Specific genotype-tumour risk associations provides a basis for novel investigative strategies into succinate dehydrogenase-related mechanisms of tumourigenesis and the development of personalised management for SDHB/SDHC/SDHD mutation carriers.

Assessing the effectiveness of NICE criteria for stratifying breast cancer risk in a UK cohort.

Breast cancer risk is a common indication for referral to clinical genetics services. UK National Institute of Health and Care Excellence (NICE) guidelines use family history (FH) to stratify by 10-year risk of breast cancer from age 40. Patients are stratified into population risk (PR, 10-year risk <3%), moderate (MR, 3-8%) and high risk (HR, >8%). Women at increased risk are offered screening at or prior to age 40. To assess the clinical effectiveness of current risk stratification, FH data were obtained for all unaffected women with a FH of breast cancer aged <50, referred to cancer genetics from 2000-2010. Patients were risk stratified by NICE criteria, identifying patients who subsequently developed breast cancer. A total of 1409 women had 15,414 patient years of follow-up. Thirty invasive breast cancers developed, 13 in MR and 13 in HR women. Kaplan-Meier analysis demonstrated no significant difference in the rate of breast cancer development between PR and MR women from ages 40 to 49 (Log rank p = 0.431). There was a significant difference between ages 40 and 49 years between PR and HR women (p = 0.036), but not on exclusion of BRCA mutation carriers (p = 0.136). NICE absolute 10-year risk thresholds between ages 40 and 49 were not met in any risk group, when risk was estimated using the guidelines (PR = 0.82%, MR = 1.68%, HR = 3.56%). Our data suggest that improved criteria are required for risk assessment prior to age 50 and screening resources may be best focussed on those with highly penetrant mutations in cancer risk genes.

Mutations specific to the Rac-GEF domain of TRIO cause intellectual disability and microcephaly.

BACKGROUND: Neurodevelopmental disorders have challenged clinical genetics for decades, with over 700 genes implicated and many whose function remains unknown. The application of whole-exome sequencing is proving pivotal in closing the genotype/phenotype gap through the discovery of new genes and variants that help to unravel the pathogenic mechanisms driving neuropathogenesis. One such discovery includes TRIO, a gene recently implicated in neurodevelopmental delay. Trio is a Dbl family guanine nucleotide exchange factor (GEF) and a major regulator of neuronal development, controlling actin cytoskeleton dynamics by activating the GTPase Rac1. METHODS: Whole-exome sequencing was undertaken on a family presenting with global developmental delay, microcephaly and mild dysmorphism. Father/daughter exome analysis was performed, followed by confirmatory Sanger sequencing and segregation analysis on four individuals. Three further patients were recruited through the deciphering developmental disorders (DDD) study. Functional studies were undertaken using patient-specific Trio protein mutations. RESULTS: We identified a frameshift deletion in TRIO that segregated autosomal dominantly. By scrutinising data from DDD, we further identified three unrelated children with a similar phenotype who harboured de novo missense mutations in TRIO. Biochemical studies demonstrated that in three out of four families, the Trio mutations led to a markedly reduced Rac1 activation. CONCLUSIONS: We describe an inherited global developmental delay phenotype associated with a frameshift deletion in TRIO. Additionally, we identify pathogenic de novo missense mutations in TRIO associated with the same consistent phenotype, intellectual disability, microcephaly and dysmorphism with striking digital features. We further functionally validate the importance of the GEF domain in Trio protein function. Our study demonstrates how genomic technologies are yet again proving prolific in diagnosing and advancing the understanding of neurodevelopmental disorders.

Genetic Analysis of 'PAX6-Negative' Individuals with Aniridia or Gillespie Syndrome.

We report molecular genetic analysis of 42 affected individuals referred with a diagnosis of aniridia who previously screened as negative for intragenic PAX6 mutations. Of these 42, the diagnoses were 31 individuals with aniridia and 11 individuals referred with a diagnosis of Gillespie syndrome (iris hypoplasia, ataxia and mild to moderate developmental delay). Array-based comparative genomic hybridization identified six whole gene deletions: four encompassing PAX6 and two encompassing FOXC1. Six deletions with plausible cis-regulatory effects were identified: five that were 3' (telomeric) to PAX6 and one within a gene desert 5' (telomeric) to PITX2. Sequence analysis of the FOXC1 and PITX2 coding regions identified two plausibly pathogenic de novo FOXC1 missense mutations (p.Pro79Thr and p.Leu101Pro). No intragenic mutations were detected in PITX2. FISH mapping in an individual with Gillespie-like syndrome with an apparently balanced X;11 reciprocal translocation revealed disruption of a gene at each breakpoint: ARHGAP6 on the X chromosome and PHF21A on chromosome 11. In the other individuals with Gillespie syndrome no mutations were identified in either of these genes, or in HCCS which lies close to the Xp breakpoint. Disruption of PHF21A has previously been implicated in the causation of intellectual disability (but not aniridia). Plausibly causative mutations were identified in 15 out of 42 individuals (12/32 aniridia; 3/11 Gillespie syndrome). Fourteen of these mutations presented in the known aniridia genes; PAX6, FOXC1 and PITX2. The large number of individuals in the cohort with no mutation identified suggests greater locus heterogeneity may exist in both isolated and syndromic aniridia than was previously appreciated.