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BackgroundPrimary ciliary dyskinesia (PCD), a genetically heterogeneous condition enriched in some consanguineous populations, results from recessive mutations affecting cilia biogenesis and motility. Currently, diagnosis requires multiple expert tests.MethodsThe diagnostic utility of multigene panel next-generation sequencing (NGS) was evaluated in 161 unrelated families from multiple population ancestries.ResultsMost (82%) families had affected individuals with biallelic or hemizygous (75%) or single (7%) pathogenic causal alleles in known PCD genes. Loss-of-function alleles dominate (73% frameshift, stop-gain, splice site), most (58%) being homozygous, even in non-consanguineous families. Although 57% (88) of the total 155 diagnostic disease variants were novel, recurrent mutations and mutated genes were detected. These differed markedly between white European (52% of families carry DNAH5 or DNAH11 mutations), Arab (42% of families carry CCDC39 or CCDC40 mutations) and South Asian (single LRRC6 or CCDC103 mutations carried in 36% of families) patients, revealing a striking genetic stratification according to population of origin in PCD. Genetics facilitated successful diagnosis of 81% of families with normal or inconclusive ultrastructure and 67% missing prior ultrastructure results.ConclusionsThis study shows the added value of high-throughput targeted NGS in expediting PCD diagnosis. Therefore, there is potential significant patient benefit in wider and/or earlier implementation of genetic screening.

Monogenic autoinflammatory diseases (AID) are a rapidly expanding group of genetically diverse but phenotypically overlapping systemic inflammatory disorders associated with dysregulated innate immunity. They cause significant morbidity, mortality and economic burden. Here, we aimed to develop and evaluate the clinical impact of a NGS targeted gene panel, the \"Vasculitis and Inflammation Panel\" (VIP) for AID and vasculitis. The Agilent SureDesign tool was used to design 2 versions of VIP; VIP1 targeting 113 genes, and a later version, VIP2, targeting 166 genes. Captured and indexed libraries (QXT Target Enrichment System) prepared for 72 patients were sequenced as a multiplex of 16 samples on an Illumina MiSeq sequencer in 150bp paired-end mode. The cohort comprised 22 positive control DNA samples from patients with previously validated mutations in a variety of the genes; and 50 prospective samples from patients with suspected AID in whom previous Sanger based genetic screening had been non-diagnostic. VIP was sensitive and specific at detecting all the different types of known mutations in 22 positive controls, including gene deletion, small INDELS, and somatic mosaicism with allele fraction as low as 3%. Six/50 patients (12%) with unclassified AID had at least one class 5 (clearly pathogenic) variant; and 11/50 (22%) had at least one likely pathogenic variant (class 4). Overall, testing with VIP resulted in a firm or strongly suspected molecular diagnosis in 16/50 patients (32%). The high diagnostic yield and accuracy of this comprehensive targeted gene panel validate the use of broad NGS-based testing for patients with suspected AID.

Glycosylphosphatidylinositol anchored proteins (GPI‐APs) represent a class of molecules attached to the external leaflet of the plasma membrane by the GPI anchor where they play important roles in numerous cellular processes including neurogenesis, cell adhesion, immune response and signalling. Within the group of GPI anchor defects, six present with the clinical phenotype of Hyperphosphatasia with Mental Retardation Syndrome (HPMRS, Mabry Syndrome) characterized by moderate to severe intellectual disability, dysmorphic features, hypotonia, seizures and persistent hyperphosphatasia. We report the case of a 5‐year‐old female with global developmental delay associated with precocious puberty and persistently raised plasma alkaline phosphatase. Targeted next generation sequencing analysis of the HPMRS genes identified novel compound heterozygous variants in the PGAP2 gene (c.103del p.(Leu35Serfs*90)and c.134A > Gp.(His45Arg)) consistent with the diagnosis of HPMRS type 3. Cerebrospinal fluid (CSF) neurotransmitter analysis showed low levels of pyridoxal phosphate and 5‐methyltetrahydrofolate and raised homovanillic acid. Supplementation with pyridoxine and folinic acid led to normalization of biochemical abnormalities. The patient continues to make developmental progress with significant improvement in speech and fine motor skills. Our reported case expands the clinical spectrum of HPMRS3 in which multisystem involvement is being increasingly recognized. Furthermore, it shows that miss‐targeting GPI‐APs and the effect on normal cellular function could provide a physiopathologic explanation for the CSF biochemical abnormalities with management implications for a group of disorders that currently has no treatment that can lead possibly to improved clinical outcomes.

Background Mitochondrial membrane protein‐associated neurodegeneration (MPAN) is caused by pathogenic sequence variants in C19orf12. Autosomal recessive inheritance has been demonstrated. We present evidence of autosomal dominant MPAN and propose a mechanism to explain these cases. Methods Two large families with apparently dominant MPAN were investigated; additional singleton cases of MPAN were identified. Gene sequencing and multiplex ligation‐dependent probe amplification were used to characterize the causative sequence variants in C19orf12. Post‐mortem brain from affected subjects was examined. Results In two multi‐generation non‐consanguineous families, we identified different nonsense sequence variations in C19orf12 that segregate with the MPAN phenotype. Brain pathology was similar to that of autosomal recessive MPAN. We additionally identified a preponderance of cases with single heterozygous pathogenic sequence variants, including two with de novo changes. Conclusions We present three lines of clinical evidence to demonstrate that MPAN can manifest as a result of only one pathogenic C19orf12 sequence variant. We propose that truncated C19orf12 proteins, resulting from nonsense variants in the final exon in our autosomal dominant cohort, impair function of the normal protein produced from the non‐mutated allele via a dominant negative mechanism and cause loss of function. These findings impact the clinical diagnostic evaluation and counseling. Autosomal recessive inheritance has been demonstrated for mitochondrial membrane protein‐associated neurodegeneration (MPAN). Here we provide 3 lines of evidence for autosomal dominant inheritance in a portion of cases: (1) a dominant inheritance pattern in 2 large, non‐consanguineous families; (2) manifestation of MPAN in 2 cases with single de novo pathogenic sequence variants; (3) a preponderance of heterozygous cases with frameshift/premature stop or nonsense variants clustering in the last exon.

Rapid technological advances in genomic testing continue to increase our understanding of the genetic basis of a wide range of kidney disorders. Establishing a molecular diagnosis benefits the individual by bringing an end to what is often a protracted diagnostic odyssey, facilitates accurate reproductive counselling for families and, in the future, is likely to lead to the delivery of more targeted management and surveillance regimens. The selection of the most appropriate testing modality requires an understanding both of the technologies available and of the genetic architecture and heterogeneity of kidney disease. Whilst we are witnessing a far greater diagnostic yield with broader genetic testing, such approaches invariably generate variants of uncertain significance and secondary incidental findings, which are not only difficult to interpret but present ethical challenges with reporting and feeding back to patients and their families. Here, we review the spectrum of nephrogenetic disorders, consider the optimal approach to genetic testing, explore the clinical utility of obtaining a molecular diagnosis, reflect on the challenges of variant interpretation and look to the future of this dynamic field.

Primary ciliary dyskinesia (PCD) is associated with abnormal organ positioning (situs) and congenital heart disease (CHD). This study investigated genotype–phenotype associations in PCD to facilitate risk predictions for cardiac and laterality defects. This retrospective cohort study of 389 UK patients with PCD found 51% had abnormal situs and 25% had CHD and/or laterality defects other than situs inversus totalis. Patients with biallelic mutations in a subset of nine PCD genes had normal situs. Patients with consanguineous parents had higher odds of situs abnormalities than patients with non-consanguineous parents. Patients with abnormal situs had higher odds of CHD and/or laterality defects.

Cenani-Lenz syndactyly (CLS) is a rare autosomal recessive syndrome characterized by disorganized oligosyndactyly of upper and lower limbs as well as radioulnar synostosis. Structural renal abnormalities are also common. We report two affected brothers, born to orthodox Jewish parents, in whom we found a novel homozygous missense variant c.4910G>A; p.(Cys1637Tyr) in LRP4 situated in an EGF-like domain between the fourth beta-propeller and transmembrane domains. Both brothers have had recurrent ketotic hypoglycaemia which has not been associated previously. We present 3D computed tomographic imaging illustrating the limb abnormalities in detail.

Heinz Jungbluth and colleagues report the identification of mutations in EPG5 that cause Vici syndrome, characterized by callosal agenesis, cataracts, cardiomyopathy, combined immunodeficiency and hypopigmentation. EPG5 encodes a regulator of autophagy, and the identified mutations cause defective autophagosomal function. Vici syndrome is a recessively inherited multisystem disorder characterized by callosal agenesis, cataracts, cardiomyopathy, combined immunodeficiency and hypopigmentation. To investigate the molecular basis of Vici syndrome, we carried out exome and Sanger sequence analysis in a cohort of 18 affected individuals. We identified recessive mutations in EPG5 (previously KIAA1632 ), indicating a causative role in Vici syndrome. EPG5 is the human homolog of the metazoan-specific autophagy gene epg-5 , encoding a key autophagy regulator (ectopic P-granules autophagy protein 5) implicated in the formation of autolysosomes. Further studies showed a severe block in autophagosomal clearance in muscle and fibroblasts from individuals with mutant EPG5 , resulting in the accumulation of autophagic cargo in autophagosomes. These findings position Vici syndrome as a paradigm of human multisystem disorders associated with defective autophagy and suggest a fundamental role of the autophagy pathway in the immune system and the anatomical and functional formation of organs such as the brain and heart.

Great Ormond Street Hospital has been a pioneer in offering Whole Genome Sequencing (WGS) to children with complex medical problems, for example through huge recruitment to the 100,000 Genome Study. Genetic technologies herald great diagnostic promise for many children and access to WGS should increase with the launch of the new NHS Genomic Medicine Service. However, interpretation of genetic data and results is challenging. We present cases of new genetic diagnoses from the 100,000 Genome Study which illustrate the revolutionary role WGS can provide in diagnosing children and identifying new genetic conditions, yet demonstrate that clinical acumen and an understanding of genetic technologies is still critical for diagnosis.Cohen syndrome is a genetic disorder characterised by intellectual impairment, developmental delay, microcephaly, and distinctive facial features, associated with homozygous alterations in the VPS13B gene. We describe two patients for whom a new diagnosis was confirmed only through a combination of clinical suspicion, microarray and WGS. Only a multi-pronged approach permitted recognition of a mono-allelic VPS13B deletion alongside a sequencing variant on the other allele.Geleophysic dysplasia is a rare skeletal dysplasia associated with heterozygous alterations in LTBP3. In-house clinical exome sequencing and WGS were both undertaken for a patient with a typical geleophysic phenotype. However, due to methodological differences, a likely pathogenic 32 base-pair duplication was only identified through one technique, emphasising the need for an appreciation of how genetic alterations can sometimes be missed without regular reappraisal, awareness of phenotype-genotype specificity, and an understanding of genomic methodology and caveats.Such cases highlight the importance of lifelong-learning in genomic medicine and why paediatricians should be aware of new developments, strengths and limitations of genetic testing. We propose that a new educational framework is required for clinicians and trainee doctors to maximise patient benefit from the new NHS Genomic Medicine Service.