The impact of inversions across 33,924 families with rare disease from a national genome sequencing project.

Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.

Systematic identification of disease-causing promoter and untranslated region variants in 8,040 undiagnosed individuals with rare disease.

Background: Both promoters and untranslated regions (UTRs) have critical regulatory roles, yet variants in these regions are largely excluded from clinical genetic testing due to difficulty in interpreting pathogenicity. The extent to which these regions may harbour diagnoses for individuals with rare disease is currently unknown. Methods: We present a framework for the identification and annotation of potentially deleterious proximal promoter and UTR variants in known dominant disease genes. We use this framework to annotate de novo variants (DNVs) in 8,040 undiagnosed individuals in the Genomics England 100,000 genomes project, which were subject to strict region-based filtering, clinical review, and validation studies where possible. In addition, we performed region and variant annotation-based burden testing in 7,862 unrelated probands against matched unaffected controls. Results: We prioritised eleven DNVs and identified an additional variant overlapping one of the eleven. Ten of these twelve variants (82%) are in genes that are a strong match to the individual's phenotype and six had not previously been identified. Through burden testing, we did not observe a significant enrichment of potentially deleterious promoter and/or UTR variants in individuals with rare disease collectively across any of our region or variant annotations. Conclusions: Overall, we demonstrate the value of screening promoters and UTRs to uncover additional diagnoses for previously undiagnosed individuals with rare disease and provide a framework for doing so without dramatically increasing interpretation burden.

A phenome-wide association study of methylated GC-rich repeats identifies a GCC repeat expansion in AFF3 as a significant cause of intellectual disability.

GC-rich tandem repeat expansions (TREs) are often associated with DNA methylation, gene silencing and folate-sensitive fragile sites and underlie several congenital and late-onset disorders. Through a combination of DNA methylation profiling and tandem repeat genotyping, we identified 24 methylated TREs and investigated their effects on human traits using PheWAS in 168,641 individuals from the UK Biobank, identifying 156 significant TRE:trait associations involving 17 different TREs. Of these, a GCC expansion in the promoter of AFF3 was linked with a 2.4-fold reduced probability of completing secondary education, an effect size comparable to several recurrent pathogenic microdeletions. In a cohort of 6,371 probands with neurodevelopmental problems of suspected genetic etiology, we observed a significant enrichment of AFF3 expansions compared to controls. With a population prevalence that is at least 5-fold higher than the TRE that causes fragile X syndrome, AFF3 expansions represent a significant cause of neurodevelopmental delay.

Genetic risk for Huntington Disease and reproductive decision-making: A systematic review.

Huntington Disease (HD) is an incurable autosomal dominant single gene neurodegenerative disorder. Typical onset is between 30 and 40 years and characterised by motor difficulties, cognitive impairment, and behavioural and personality changes. The availability of reproductive testing means that affected and at-risk individuals can make reproductive decisions with genetic risk in mind. We aimed to summarise the literature on reproductive decision-making in the context of HD risk in terms of outcomes and the subjective experiences of at-risk individuals. Five databases were searched. Findings were synthesised using Framework analysis to identify common factors across results of quantitative and qualitative studies. Twenty five studies met inclusion criteria. Framework analysis identified the following key areas: 'The relationship between reproductive intentions and HD genetic risk', 'Views on assistive options', 'Complexity and challenges in reproductive decision-making', 'Actual reproductive outcomes', and 'Other factors influencing reproductive decision-making'. Quality of included studies was mixed. Reproductive decision making in the context of HD risk was found to be a complex and emotionally challenging process. Further research is required into reproductive decision-making and outcomes among those not utilising assistive options, and in developing a model of reproductive decision-making in HD.

Amniotic band sequence in vascular Ehlers-Danlos Syndrome (EDS): Experience of the EDS National Diagnostic Services in the UK.

The association between vascular Ehlers-Danlos Syndrome (vEDS) and amniotic band sequence (ABS) has been previously reported in the literature, mostly in single patient case reports. Here, we aim to extend the current knowledge of this association through a case series of five unrelated individuals with ABS in association with molecularly confirmed vEDS, in addition to undertaking a comprehensive literature review. All the individuals were recruited through the EDS national diagnostic service in the UK following appropriate history, physical examination and genetic investigations. Clinical presentation ranged from a single constriction ring to complex craniofacial clefts to limb reduction deformities, reflecting the spectrum of ABS presentation. vEDS was inherited paternally (n = 2), maternally (n = 2) and de novo (n = 1). Previously, maternal vEDS was considered the risk factor for ABS, but our findings suggest that it may be the disease status of the fetus which poses the main risk. It is established that amniotic membrane is derived from fetal tissue, which supports our conclusions. Our observations suggest the increased risk of ABS in fetuses with vEDS. Therefore, exploring family history and features that may suggest vEDS diagnosis in patients with ABS might be useful. We also recommend that a collaborative international study would be useful to help gain a better insight into this association.

Natural history of NF1 c.2970_2972del p.(Met992del): confirmation of a low risk of complications in a longitudinal study.

Individuals with the three base pair deletion NM_000267.3(NF1):c.2970_2972del p.(Met992del) have been recognised to present with a milder neurofibromatosis type 1 (NF1) phenotype characterised by café-au-lait macules (CALs) and intertriginous freckling, as well as a lack of cutaneous, subcutaneous and plexiform neurofibromas and other NF1-associated complications. Examining large cohorts of patients over time with this specific genotype is important to confirm the presentation and associated risks of this variant across the lifespan. Forty-one individuals with the in-frame NF1 deletion p.Met992del were identified from 31 families. Clinicians completed a standardised clinical questionnaire for each patient and the resulting data were collated and compared to published cohorts. Thirteen patients have been previously reported, and updated clinical information has been obtained for these individuals. Both CALs and intertriginous freckling were present in the majority of individuals (26/41, 63%) and the only confirmed features in 11 (27%). 34/41 (83%) of the cohort met NIH diagnostic criteria. There was a notable absence of all NF1-associated tumour types (neurofibroma and glioma). Neurofibroma were observed in only one individual-a subcutaneous lesion (confirmed histologically). Nineteen individuals were described as having a learning disability (46%). This study confirms that individuals with p.Met992del display a mild tumoural phenotype compared to those with 'classical', clinically diagnosed NF1, and this appears to be the case longitudinally through time as well as at presentation. Learning difficulties, however, appear to affect a significant proportion of NF1 subjects with this phenotype. Knowledge of this genotype-phenotype association is fundamental to accurate prognostication for families and caregivers.

First presentation of LPIN1 acute rhabdomyolysis in adolescence and adulthood.

LPIN1 mutations are a known common cause of autosomal recessive, recurrent and life-threatening acute rhabdomyolysis of childhood-onset. The first episode of rhabdomyolysis usually happens in nearly all cases before the age of 5 and death is observed in 1/3 of patients. Here we present two cases of acute rhabdomyolysis with a milder phenotype caused by LPIN1 mutation presenting in adolescence (11 years old) and adulthood (40 years old) after Parvovirus infection and metabolic stress, respectively. In our opinion, the mutation types, epigenetic factors, the environment exposition to triggers or the existence of proteins with a similar structure of LPIN1, may have a role in modulating the onset of rhabdomyolysis. LPIN1 should be included on a panel of genes analysed in the investigation of adult individuals with rhabdomyolysis. Metabolic and viral stressors should be included in the list of possible rhabdomyolysis precipitant.

Multi-level evidence of an allelic hierarchy of USH2A variants in hearing, auditory processing and speech/language outcomes.

Language development builds upon a complex network of interacting subservient systems. It therefore follows that variations in, and subclinical disruptions of, these systems may have secondary effects on emergent language. In this paper, we consider the relationship between genetic variants, hearing, auditory processing and language development. We employ whole genome sequencing in a discovery family to target association and gene x environment interaction analyses in two large population cohorts; the Avon Longitudinal Study of Parents and Children (ALSPAC) and UK10K. These investigations indicate that USH2A variants are associated with altered low-frequency sound perception which, in turn, increases the risk of developmental language disorder. We further show that Ush2a heterozygote mice have low-level hearing impairments, persistent higher-order acoustic processing deficits and altered vocalizations. These findings provide new insights into the complexity of genetic mechanisms serving language development and disorders and the relationships between developmental auditory and neural systems.

Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism.

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified. Analysis of the breakpoint junction fragment indicated that these 67 kb heterozygous duplications were likely mediated by non-allelic homologous recombination at regions of high sequence identity in ATAD3A exon 11 and ATAD3C exon 7. At the recombinant junction, the duplication allele produces a fusion gene derived from ATAD3A and ATAD3C, the protein product of which lacks key functional residues. Analysis of fibroblasts derived from two affected individuals shows that the fusion gene product is expressed and stable. These cells display perturbed cholesterol and mitochondrial DNA organization similar to that observed for individuals with severe ATAD3A deficiency. We hypothesize that the fusion protein acts through a dominant-negative mechanism to cause this fatal mitochondrial disorder. Our data delineate a molecular diagnosis for this disorder, extend the clinical spectrum associated with structural variation at the ATAD3 locus, and identify a third mutational mechanism for ATAD3 gene cluster variants. These results further affirm structural variant mutagenesis mechanisms in sporadic disease traits, emphasize the importance of copy number analysis in molecular genomic diagnosis, and highlight some of the challenges of detecting and interpreting clinically relevant rare gene rearrangements from next-generation sequencing data.

Xq27.1 Duplication Encompassing SOX3: Variable Phenotype and Smallest Duplication Associated with Hypopituitarism to Date - A Large Case Series of Unrelated Patients and a Literature Review.

BACKGROUND: Xq27.1 duplication encompassing SOX3 has been implicated in the aetiology of X-linked hypopituitarism associated with intellectual disability and neural tube defects. We describe the largest case series to date of 5 unrelated patients with SOX3 duplication with a variable clinical phenotype, including the smallest reported SOX3 duplication. CASE REPORTS: Five male patients who presented with congenital hypopituitarism (CH) were identified to have Xq27.1 duplication encompassing SOX3. The size of the duplication ranged from 323.8 kb to 11 Mb. The duplication was maternally inherited or de novo in 2 patients each (and of unknown inheritance in 1 patient). The age at presentation was variable. Three patients had multiple pituitary hormone deficiencies, whereas 2 patients had isolated growth hormone deficiency. All patients had micropenis and/or small undescended testes. Structural pituitary and/or other midline cranial abnormalities (callosal hypogenesis/absence of the septum pellucidum) were present in all patients. Two patients had a neural tube defect in addition to CH. CONCLUSIONS: This is the largest series reported to date of unrelated patients with CH in association with Xq27.1 duplication encompassing SOX3. The clinical phenotype is variable, which may be due to genetic redundancy or other unknown aetiological factors. We have expanded the phenotypic spectrum through description of the smallest Xq27.1 duplication (323.8 kb) with CH reported to date, as well as a second family with CH and a neural tube defect.

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.

Delineation of dominant and recessive forms of LZTR1-associated Noonan syndrome.

Noonan syndrome (NS) is characterised by distinctive facial features, heart defects, variable degrees of intellectual disability and other phenotypic manifestations. Although the mode of inheritance is typically dominant, recent studies indicate LZTR1 may be associated with both dominant and recessive forms. Seeking to describe the phenotypic characteristics of LZTR1-associated NS, we searched for likely pathogenic variants using two approaches. First, scrutiny of exomes from 9624 patients recruited by the Deciphering Developmental Disorders (DDDs) study uncovered six dominantly-acting mutations (p.R97L; p.Y136C; p.Y136H, p.N145I, p.S244C; p.G248R) of which five arose de novo, and three patients with compound-heterozygous variants (p.R210*/p.V579M; p.R210*/p.D531N; c.1149+1G>T/p.R688C). One patient also had biallelic loss-of-function mutations in NEB, consistent with a composite phenotype. After removing this complex case, analysis of human phenotype ontology terms indicated significant phenotypic similarities (P = 0.0005), supporting a causal role for LZTR1. Second, targeted sequencing of eight unsolved NS-like cases identified biallelic LZTR1 variants in three further subjects (p.W469*/p.Y749C, p.W437*/c.-38T>A and p.A461D/p.I462T). Our study strengthens the association of LZTR1 with NS, with de novo mutations clustering around the KT1-4 domains. Although LZTR1 variants explain ~0.1% of cases across the DDD cohort, the gene is a relatively common cause of unsolved NS cases where recessive inheritance is suspected.

De novo and biallelic DEAF1 variants cause a phenotypic spectrum.

PURPOSE: To investigate the effect of different DEAF1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and on DEAF1 activity in vitro. METHODS: We assembled a cohort of 23 patients with de novo and biallelic DEAF1 variants, described the genotype-phenotype correlation, and investigated the differential effect of de novo and recessive variants on transcription assays using DEAF1 and Eif4g3 promoter luciferase constructs. RESULTS: The proportion of the most prevalent phenotypic features, including intellectual disability, speech delay, motor delay, autism, sleep disturbances, and a high pain threshold, were not significantly different in patients with biallelic and pathogenic de novo DEAF1 variants. However, microcephaly was exclusively observed in patients with recessive variants (p < 0.0001). CONCLUSION: We propose that different variants in the DEAF1 gene result in a phenotypic spectrum centered around neurodevelopmental delay. While a pathogenic de novo dominant variant would also incapacitate the product of the wild-type allele and result in a dominant-negative effect, a combination of two recessive variants would result in a partial loss of function. Because the clinical picture can be nonspecific, detailed phenotype information, segregation, and functional analysis are fundamental to determine the pathogenicity of novel variants and to improve the care of these patients.

Duplication of 10q24 locus: broadening the clinical and radiological spectrum.

Split-hand-split-foot malformation (SHFM) is a rare condition that occurs in 1 in 8500-25,000 newborns and accounts for 15% of all limb reduction defects. SHFM is heterogeneous and can be isolated, associated with other malformations, or syndromic. The mode of inheritance is mostly autosomal dominant with incomplete penetrance, but can be X-linked or autosomal recessive. Seven loci are currently known: SHFM1 at 7q21.2q22.1 (DLX5 gene), SHFM2 at Xq26, SHFM3 at 10q24q25, SHFM4 at 3q27 (TP63 gene), SHFM5 at 2q31 and SHFM6 as a result of variants in WNT10B (chromosome 12q13). Duplications at 17p13.3 are seen in SHFM when isolated or associated with long bone deficiency. Tandem genomic duplications at chromosome 10q24 involving at least the DACTYLIN gene are associated with SHFM3. No point variant in any of the genes residing within the region has been identified so far, but duplication of exon 1 of the BTRC gene may explain the phenotype, with likely complex alterations of gene regulation mechanisms that would impair limb morphogenesis. We report on 32 new index cases identified by array-CGH and/or by qPCR, including some prenatal ones, leading to termination for the most severe. Twenty-two cases were presenting with SHFM and 7 with monodactyly only. Three had an overlapping phenotype. Additional findings were identified in 5 (renal dysplasia, cutis aplasia, hypogonadism and agenesis of corpus callosum with hydrocephalus). We present their clinical and radiological findings and review the literature on this rearrangement that seems to be one of the most frequent cause of SHFM.

27 years of prenatal diagnosis for Huntington disease in the United Kingdom.

PURPOSE: There is little long-term, population-based data on uptake of prenatal diagnosis for Huntington disease (HD), a late-onset autosomal dominant neurodegenerative disorder, and the effect of the availability of preimplantation genetic diagnosis (PGD) on families' decisions about conventional prenatal diagnosis is not known. We report trends in prenatal diagnosis and preimplantation diagnosis for HD in the United Kingdom since services commenced. METHODS: Long-term UK-wide prospective case record-based service evaluation in 23 UK Regional Genetic Centres 1988-2015, and four UK PGD centers 2002-2015. RESULTS: From 1988 to 2015, 479 prenatal diagnoses were performed in the UK for HD. An exclusion approach was used in 150 (31%). The annual rate of HD prenatal diagnosis has remained around 18 (3.5/million) over 27 years, despite a steady increase in the use of PGD for HD since 2002. CONCLUSION: Although increasing number of couples are choosing either direct or exclusion PGD to prevent HD in their offspring, both direct and exclusion prenatal diagnosis remain important options in a health system where both PGD and prenatal diagnosis are state funded. At-risk couples should be informed of all options available to them, preferably prepregnancy.

Predictive testing of minors for Huntington's disease: The UK and Netherlands experiences.

A consistent feature of predictive testing guidelines for Huntington's disease (HD) is the recommendation not to undertake predictive tests on those < 18 years. Exceptions are made but the extent of, and reasons for, deviation from the guidelines are unknown. The UK Huntington's Prediction Consortium has collected data annually on predictive tests undertaken from the 23 UK genetic centers. DNA analysis for HD in the Netherlands is centralized in the Laboratory for Diagnostic Genome Analysis in Leiden. In the UK, 60 tests were performed on minors between 1994 and 2015 representing 0.63% of the total number of tests performed. In the Netherlands, 23 tests were performed on minors between 1997 and 2016. The majority of the tests were performed on those aged 16 and 17 years for both countries (23% and 57% for the UK, and 26% and 57% for the Netherlands). Data on the reasons for testing were identified for 36 UK and 22 Netherlands cases and included: close to the age of 18 years, pregnancy, currently in local authority care and likely to have less support available after 18 years, person never having the capacity to consent and other miscellaneous reasons. This study documents the extent of HD testing of minors in the UK and the Netherlands and suggests that, in general, the recommendation is being followed. We provide some empirical evidence as to reasons why clinicians have departed from the recommendation. We do not advise changing the recommendation but suggest that testing of minors continues to be monitored.

A cohort of 17 patients with kyphoscoliotic Ehlers-Danlos syndrome caused by biallelic mutations in FKBP14: expansion of the clinical and mutational spectrum and description of the natural history.

PurposeIn 2012 we reported in six individuals a clinical condition almost indistinguishable from PLOD1-kyphoscoliotic Ehlers-Danlos syndrome (PLOD1-kEDS), caused by biallelic mutations in FKBP14, and characterized by progressive kyphoscoliosis, myopathy, and hearing loss in addition to connective tissue abnormalities such as joint hypermobility and hyperelastic skin. FKBP14 is an ER-resident protein belonging to the family of FK506-binding peptidyl-prolyl cis-trans isomerases (PPIases); it catalyzes the folding of type III collagen and interacts with type III, type VI, and type X collagens. Only nine affected individuals have been reported to date.MethodsWe report on a cohort of 17 individuals with FKBP14-kEDS and the follow-up of three previously reported patients, and provide an extensive overview of the disorder and its natural history based on clinical, biochemical, and molecular genetics data.ResultsBased on the frequency of the clinical features of 23 patients from the present and previous cohorts, we define major and minor features of FKBP14-kEDS. We show that myopathy is confirmed by histology and muscle imaging only in some patients, and that hearing impairment is predominantly sensorineural and may not be present in all individuals.ConclusionOur data further support the extensive clinical overlap with PLOD1-kEDS and show that vascular complications are rare manifestations of FKBP14-kEDS.

Increased central microglial activation associated with peripheral cytokine levels in premanifest Huntington's disease gene carriers.

Previous studies have shown activation of the immune system and altered immune response in Huntington's disease (HD) gene carriers. Here, we hypothesized that peripheral and central immune responses could be concurrent pathophysiological events and represent a global innate immune response to the toxic effects of mutant huntingtin in HD gene carriers. We sought to investigate our hypothesis using [(11)C]PK11195 PET as a translocator protein (TSPO) marker of central microglial activation, together with assessment of peripheral plasma cytokine levels in a cohort of premanifest HD gene carriers who were more than a decade from predicted symptomatic conversion. Data were also compared to those from a group of healthy controls matched for age and gender. We found significantly increased peripheral plasma IL-1ß levels in premanifest HD gene carriers compared to the group of normal controls (P=0.018). Premanifest HD gene carriers had increased TSPO levels in cortical, basal ganglia and thalamic brain regions (P<0.001). Increased microglial activation in somatosensory cortex correlated with higher plasma levels of IL-1ß (rs=0.87, P=0.013), IL-6 (rs=0.85, P=0.013), IL-8 (rs=0.68, P=0.045) and TNF-α (rs=0.79; P=0.013). Our findings provide first in vivo evidence for an association between peripheral and central immune responses in premanifest HD gene carriers, and provide further supporting evidence for the role of immune dysfunction in the pathogenesis of HD.