Systematic reanalysis of copy number losses of uncertain clinical significance.

BACKGROUND: Reanalysis of exome/genome data improves diagnostic yield. However, the value of reanalysis of clinical array comparative genomic hybridisation (aCGH) data has never been investigated. Case-by-case reanalysis can be challenging in busy diagnostic laboratories. METHODS AND RESULTS: We harmonised historical postnatal clinical aCGH results from ~16 000 patients tested via our diagnostic laboratory over ~7 years with current clinical guidance. This led to identification of 37 009 copy number losses (CNLs) including 33 857 benign, 2173 of uncertain significance and 979 pathogenic. We found benign CNLs to be significantly less likely to encompass haploinsufficient genes compared with the pathogenic or CNLs of uncertain significance in our database. Based on this observation, we developed a reanalysis pipeline using up-to-date disease association data and haploinsufficiency scores and shortlisted 207 CNLs of uncertain significance encompassing at least one autosomal dominant disease-gene associated with haploinsufficiency or loss-of-function mechanism. Clinical scientist reviews led to reclassification of 15 CNLs of uncertain significance as pathogenic or likely pathogenic. This was ~0.7% of the starting cohort of 2173 CNLs of uncertain significance and 7.2% of 207 shortlisted CNLs. The reclassified CNLs included first cases of CNV-mediated disease for some genes where all previously described cases involved only point variants. Interestingly, some CNLs could not be reclassified because the phenotypes of patients with CNLs seemed distinct from the known clinical features resulting from point variants, thus raising questions about accepted underlying disease mechanisms. CONCLUSIONS: Reanalysis of clinical aCGH data increases diagnostic yield.

Anticipatory banking of samples enables diagnosis of adenylosuccinase deficiency following molecular autopsy in an infant with vacuolating leukoencephalopathy.

Adenylosuccinase deficiency is a rare inborn error of metabolism. We present a newborn who died at 52 days of age with clinical features suggestive of severe epileptic encephalopathy and leukodystrophy of unknown cause. Post-mortem examination showed an unusual vacuolar appearance of the brain. A molecular autopsy performed via singleton clinical exome analysis revealed a known pathogenic and a variant of uncertain significance in ADSL that encodes adenylosuccinase. Tests on previously stored plasma samples showed elevated succinyladenosine and succinylaminoimidazole carboxamide riboside levels. Adenylosuccinase activity in stored fibroblasts was only ~5% of control confirming the diagnosis of adenylosuccinase deficiency in the child. The parents opted for a chorionic villus biopsy in a subsequent pregnancy and had a child unaffected by adenylosuccinase deficiency. This report adds vacuolating leukodystrophy as a novel feature of adenylosuccinase deficiency and shows the power of biochemical investigations directed by genomic studies to achieve accurate diagnosis. Importantly, this case demonstrates the importance of anticipatory banking of biological samples for reverse biochemical phenotyping in individuals with undiagnosed disorders who may not survive.

Personalised virtual gene panels reduce interpretation workload and maintain diagnostic rates of proband-only clinical exome sequencing for rare disorders.

PURPOSE: The increased adoption of genomic strategies in the clinic makes it imperative for diagnostic laboratories to improve the efficiency of variant interpretation. Clinical exome sequencing (CES) is becoming a valuable diagnostic tool, capable of meeting the diagnostic demand imposed by the vast array of different rare monogenic disorders. We have assessed a clinician-led and phenotype-based approach for virtual gene panel generation for analysis of targeted CES in patients with rare disease in a single institution. METHODS: Retrospective survey of 400 consecutive cases presumed by clinicians to have rare monogenic disorders, referred on singleton basis for targeted CES. We evaluated diagnostic yield and variant workload to characterise the usefulness of a clinician-led approach for generation of virtual gene panels that can incorporate up to three different phenotype-driven gene selection methods. RESULTS: Abnormalities of the nervous system (54.5%), including intellectual disability, head and neck (19%), skeletal system (16%), ear (15%) and eye (15%) were the most common clinical features reported in referrals. Combined phenotype-driven strategies for virtual gene panel generation were used in 57% of cases. On average, 7.3 variants (median=5) per case were retained for clinical interpretation. The overall diagnostic rate of proband-only CES using personalised phenotype-driven virtual gene panels was 24%. CONCLUSIONS: Our results show that personalised virtual gene panels are a cost-effective approach for variant analysis of CES, maintaining diagnostic yield and optimising the use of resources for clinical genomic sequencing in the clinic.

Small 6q16.1 Deletions Encompassing POU3F2 Cause Susceptibility to Obesity and Variable Developmental Delay with Intellectual Disability.

Genetic studies of intellectual disability and identification of monogenic causes of obesity in humans have made immense contribution toward the understanding of the brain and control of body mass. The leptin > melanocortin > SIM1 pathway is dysregulated in multiple monogenic human obesity syndromes but its downstream targets are still unknown. In ten individuals from six families, with overlapping 6q16.1 deletions, we describe a disorder of variable developmental delay, intellectual disability, and susceptibility to obesity and hyperphagia. The 6q16.1 deletions segregated with the phenotype in multiplex families and were shown to be de novo in four families, and there was dramatic phenotypic overlap among affected individuals who were independently ascertained without bias from clinical features. Analysis of the deletions revealed a ∼350 kb critical region on chromosome 6q16.1 that encompasses a gene for proneuronal transcription factor POU3F2, which is important for hypothalamic development and function. Using morpholino and mutant zebrafish models, we show that POU3F2 lies downstream of SIM1 and controls oxytocin expression in the hypothalamic neuroendocrine preoptic area. We show that this finding is consistent with the expression patterns of POU3F2 and related genes in the human brain. Our work helps to further delineate the neuro-endocrine control of energy balance/body mass and demonstrates that this molecular pathway is conserved across multiple species.

A maternally inherited frameshift CDKL5 variant in a male with global developmental delay and late-onset generalized epilepsy.

Pathogenic CDKL5 variants cause an X-linked dominant infantile epileptic encephalopathy, predominantly in females. This condition is characterized by an early-onset severe mixed seizure disorder. We present a maternally inherited frameshift CDKL5 c.2809_2810insA p.(Cys937Ter) variant in a 13-year-old male with severe intellectual disability and late-onset generalized epilepsy. Interestingly, the variant segregation in the family is consistent with an X-linked recessive inheritance pattern, which has not previously been described with this gene. This variant is expected to result in truncation of some CDKL5 transcripts, which could potentially account for the later seizure onset and atypical inheritance pattern. Though the possibility of this variant not being causal cannot be completely excluded, this case adds to the variability of the documented phenotypic profile and to the debate around the role of C-terminus variants in CDKL5-related disease.

An evaluation of the challenges to developing tumor BRCA1 and BRCA2 testing methodologies for clinical practice.

Ovarian cancer patients with germline or somatic pathogenic variants benefit from treatment with poly ADP ribose polymerase (PARP) inhibitors. Tumor BRCA1/2 testing is more challenging than germline testing as the majority of samples are formalin-fixed paraffin embedded (FFPE), the tumor genome is complex, and the allelic fraction of somatic variants can be low. We collaborated with 10 laboratories testing BRCA1/2 in tumors to compare different approaches to identify clinically important variants within FFPE tumor DNA samples. This was not a proficiency study but an inter-laboratory comparison to identify common issues. Each laboratory received the same tumor DNA samples ranging in genotype, quantity, quality, and variant allele frequency (VAF). Each laboratory performed their preferred next-generation sequencing method to report on the variants. No false positive results were reported in this small study and the majority of methods detected the low VAF variants. A number of variants were not detected due to the bioinformatics analysis, variant classification, or insufficient DNA. The use of hybridization capture or short amplicon methods are recommended based on a bioinformatic assessment of the data. The study highlights the importance of establishing standards and standardization for tBRCA testing particularly when the test results dictate clinical decisions regarding life extending therapies.

ERBB4 exonic deletions on chromosome 2q34 in patients with intellectual disability or epilepsy.

ERBB4 encodes the tyrosine kinase receptor HER4, a critical regulator of normal cell function and neurodevelopmental processes in the brain. One of the key ligands of HER4 is neureglin-1 (NRG1), and the HER4-NRG1 signalling pathway is essential in neural crest cell migration, and neuronal differentiation. Pharmacological inactivation of HER4 has been shown to hasten the progression of epileptogenesis in rodent models, and heterozygous ERBB4 null mice are shown to have cognitive deficits and delayed motor development. Thus far there is only a single case report in the literature of a heterozygous ERBB4 deletion in a patient with intellectual disability (ID). We identified nine subjects from five unrelated families with chromosome 2q34 deletions, resulting in heterozygous intragenic loss of multiple exons of ERBB4, associated with either non-syndromic ID or generalised epilepsy. In one family, the deletion segregated with ID in five affected relatives. Overall, this case series further supports that haploinsufficiency of ERBB4 leads to non-syndromic intellectual disability or epilepsy.

Presynaptic congenital myasthenic syndrome due to three novel mutations in SLC5A7 encoding the sodium-dependant high-affinity choline transporter.

SLC5A7 encodes the presynaptic sodium-dependant high-affinity choline transporter 1 (CHT), which uptakes choline to the presynaptic nerve terminal following the breakdown of acetylcholine by the acetylcholinesterase within the synaptic cleft. We report 5 patients from three consanguineous families with congenital myasthenic syndrome type 20 caused by novel mutations in SLC5A7. The individuals from family 1 and 2 were homozygous for c.320G>A; (p.Arg107His) and c.886G>A (p.Ala296Thr), respectively, and their phenotype was characterised by recurrent apnoeic attacks early after birth and learning and speech difficulties in childhood. Individuals from family 3 were homozygous for c.1240T>A (p.Tyr414Asn) and suffered from more severe central and peripheral manifestations with lack of spontaneous movements and respiratory drive and overall minimal response to external stimuli. All individuals tested showed neurophysiological defects compatible with impaired neuromuscular transmission. Combined treatment with cholinesterase inhibitors and ß2-adrenergic agonists was beneficial in patients from family 1 and 2. Affected individuals from family 3 died from complications directly related to their underlying genetic condition. This report provides three novel pathogenic variants in SLC5A7 and highlights the variability in the clinical phenotype, severity and prognosis of this syndrome.

Severe type I interferonopathy and unrestrained interferon signaling due to a homozygous germline mutation in STAT2.

Excessive type I interferon (IFNα/ß) activity is implicated in a spectrum of human disease, yet its direct role remains to be conclusively proven. We investigated two siblings with severe early-onset autoinflammatory disease and an elevated IFN signature. Whole-exome sequencing revealed a shared homozygous missense Arg148Trp variant in STAT2, a transcription factor that functions exclusively downstream of innate IFNs. Cells bearing STAT2R148W in homozygosity (but not heterozygosity) were hypersensitive to IFNα/ß, which manifest as prolonged Janus kinase-signal transducers and activators of transcription (STAT) signaling and transcriptional activation. We show that this gain of IFN activity results from the failure of mutant STAT2R148W to interact with ubiquitin-specific protease 18, a key STAT2-dependent negative regulator of IFNα/ß signaling. These observations reveal an essential in vivo function of STAT2 in the regulation of human IFNα/ß signaling, providing concrete evidence of the serious pathological consequences of unrestrained IFNα/ß activity and supporting efforts to target this pathway therapeutically in IFN-associated disease.

High quality clinical grade human embryonic stem cell lines derived from fresh discarded embryos.

BACKGROUND: Human embryonic stem cells (hESCs) hold tremendous promise for cell replacement therapies for a range of degenerative diseases. In order to provide cost-effective treatments affordable by public health systems, HLA-matched allogeneic tissue banks of the highest quality clinical-grade hESCs will be required. However only a small number of existing hESC lines are suitable for clinical use; they are limited by moral and ethical concerns and none of them apply Good Manufacturing Practice (GMP) standards to the earliest and critical stages of gamete and embryo procurement. We thus aimed to derive new clinical grade hESC lines of highest quality from fresh surplus GMP grade human embryos. METHODS: A comprehensive screen was performed for suitable combinations of culture media with supporting feeder cells or feeder-free matrix, at different stages, to support expansion of the inner cell mass and to establish new hESC lines. RESULTS: We developed a novel two-step and sequential media system of clinical-grade hESC derivation and successfully generated seven new hESC lines of widely varying HLA type, carefully screened for genetic health, from human embryos donated under the highest ethical and moral standards under an integrated GMP system which extends from hESC banking all the way back to gamete and embryo procurement. CONCLUSIONS: The present study, for the first time, reports the successful derivation of highest-quality clinical-grade hESC lines from fresh poor-quality surplus human embryos generated in a GMP-grade IVF laboratory. The availability of hESC lines of this status represents an important step towards more widespread application of regenerative medicine therapies.

Oculo-auriculo-vertebral spectrum: clinical and molecular analysis of 51 patients.

INTRODUCTION: Oculo-auriculo-vertebral spectrum (OAVS OMIM 164210) is a craniofacial developmental disorder affecting the development of the structures derived from the 1st and the 2nd branchial arches during embryogenesis, with consequential maxillary, mandibular, and ear abnormalities. The phenotype in OAVS is variable and associated clinical features can involve the cardiac, renal, skeletal, and central nervous systems. Its aetiology is still poorly understood. METHODS: We have evaluated the clinical phenotypes of 51 previously unpublished patients with OAVS and their parents, and performed comparative genomic hybridization microarray studies to identify potential causative loci. RESULTS: Of all 51 patients, 16 (31%) had a family history of OAVS. Most had no relevant pre-natal history and only 5 (10%) cases had a history of environmental exposures that have previously been described as risk factors for OAVS. In 28 (55%) cases, the malformations were unilateral. When the involvement was bilateral, it was asymmetric. Ear abnormalities were present in 47 (92%) patients (unilateral in 24; and bilateral in 23). Hearing loss was common (85%), mostly conductive, but also sensorineural, or a combination of both. Hemifacial microsomia was present in 46 (90%) patients (17 also presented facial nerve palsy). Ocular anomalies were present in 15 (29%) patients. Vertebral anomalies were confirmed in 10 (20%) cases; 50% of those had additional heart, brain and/or other organ abnormalities. Brain abnormalities were present in 5 (10%) patients; developmental delay was more common among these patients. Limb abnormalities were found in 6 (12%) patients, and urogenital anomalies in 5 (10%). Array-CGH analysis identified 22q11 dosage anomalies in 10 out of 22 index cases screened. DISCUSSION: In this study we carried out in-depth phenotyping of OAVS in a large, multicentre cohort. Clinical characteristics are in line with those reported previously, however, we observed a higher incidence of hemifacial microsomia and lower incidence of ocular anomalies. Furthermore our data suggests that OAVS patients with vertebral anomalies or congenital heart defects have a higher frequency of additional brain, limb or other malformations. We had a higher rate of familial cases in our cohort in comparison with previous reports, possibly because these cases were referred preferentially to our genetic clinic where family members underwent examination. We propose that familial OAVS cases show phenotypic variability, hence, affected relatives might have been misclassified in previous reports. Moreover, in view of its phenotypic variability, OAVS is potentially a spectrum of conditions, which overlap with other conditions, such as mandibulofacial dysostosis. Array CGH in our cohort identified recurrent dosage anomalies on 22q11, which may contribute to, or increase the risk of OAVS. We hypothesize that although the 22q11 locus may harbour gene(s) or regulatory elements that play a role in the regulation of craniofacial symmetry and 1st and 2nd branchial arch development, OAVS is a heterogeneous condition and many cases have a multifactorial aetiology or are caused by mutations in as yet unidentified gene(s).