Abstract Familial progressive hyper- and hypopigmentation (FPHH) is a rare autosomal dominant condition caused by pathogenic variants in the KIT ligand gene (KITLG). Here, we report an 8-year-old girl who was noted in infancy to have an increasing number of café au lait patches. On examination, she also had multiple macular depigmented lesions. Her initial presentation was thought likely to represent neurofibromatosis type 1; however, no abnormalities were detected in NF1, SPRED1 or mismatch repair genes (MLH1, MSH2, MSH6, PMS2). Further investigation of 79 genes associated with pigmentary disorders identified a variant in KITLG, c.325G>A, p.Asp109Asn, not previously reported in association with FPHH. Parental testing confirmed this was a de novo variant. Following multidisciplinary team discussion, this variant was considered highly likely to be disease causing. Our patient has since developed further pigmentary changes, with no other medical issues, including extracutaneous involvement or hearing loss. To date, 10 pathogenic variants in KITLG have been associated with FPHH. Most lie within a beta-strand in the N-terminal domain of stem cell factor (SCF), which interacts with the c-KIT receptor tyrosine kinase. The variant identified in our patient lies adjacent to this beta-strand. Additional KITLG mutations are associated with Waardenburg syndrome and nonsyndromic hearing loss. While pathogenic variants causing FPHH are thought to enhance SCF activity, leading to increased melanocyte production and survival, the mechanism underlying hypo­pigmentation remains unclear. No fertility issues or other extracutaneous manifestations have been reported in association with FPHH. This report broadens the spectrum of mutations associated with FPHH. In this case, molecular diagnosis positively influenced management by clarifying reproductive and familial risk. The presentation in our patient suggests that FPHH may be underdiagnosed and should be considered in individuals investigated for NF1 presenting with early-onset pigmentary mosaicism.

BACKGROUND:Extensive or atypical dermal melanocytosis (EDM) is abnormal macular blue/black skin pigmentation that can be associated with other congenital abnormalities and an increased risk of melanoma. It is probably underdiagnosed due to phenotypic overlap with common transient dermal melanocytosis (so-called 'blue spots'), and is frequently misdiagnosed as congenital melanocytic naevi. The genetic cause has remained largely unknown, with mosaic variants in only one gene, GNAQ, described in only a handful of cases to date. OBJECTIVES:To improve our understanding of EDM by combining deep phenotyping with genetic investigations in a large paediatric cohort. METHODS:Forty-seven patients with EDM only were recruited for phenotypic and targeted deep next-generation sequencing of affected skin. Ophthalmological examination was performed routinely, and magnetic resonance imaging (MRI) of the central nervous system (CNS) performed in five patients where there was clinical suspicion of neurological abnormalities. RESULTS:Ophthalmological involvement was seen in 40% (n = 19/47) of patients. Importantly, this was not restricted to those with periocular cutaneous involvement. Two of five patients who underwent MRI had evidence of ischaemic infarcts. Previously undescribed mosaic causes of EDM were found in HRAS, ACTB, PIK3CA and GNA11, alongside further cases of mosaic GNAQ. The HRAS variant has previously been reported in the germline causing Costello and Noonan syndromes. CONCLUSIONS:Clinically, our findings highlight the importance of differentiating EDM from common blue spots, and we recommend ophthalmological investigation even in the absence of periocular cutaneous involvement. The association with CNS infarct is unclear, but we suggest clinical neurological features be sought and MRI performed if there are concerns. Genetically, these results not only expand the causative genotypes of EDM, but also challenge our concept of the mosaic disorders currently described in association with HRAS, ACTB, PIK3CA and GNA11 variants. Stratification by genotype may help determine patient melanoma risk more accurately in the future. Patient- and variant-specific genetic counselling should be given where there is a potential risk of germline transmission to offspring as for this HRAS variant.

PURPOSE:Congenital cataracts are a leading cause of childhood blindness, with clinical impact due both to visual impairment and associated systemic disorders. They exhibit clinical and genetic heterogeneity, with pathogenic variants identified in more than 100 genes complicating genotype-phenotype correlation. Accurate molecular diagnosis is essential for prognosis, reproductive counseling, and tailored surveillance and management. Whole genome sequencing (WGS) offers advantages over gene panels by detecting a broader spectrum of variants, including copy number changes, structural rearrangements, cryptic variants, and those in repetitive regions, thereby improving diagnostic yield, especially when interpreted through multidisciplinary team meetings (MDTMs). METHODS:A retrospective review was performed of 119 consecutive patients undergoing genetic testing for congenital cataracts in the North Thames Genomic Laboratory Hub. Genetic analysis was performed using either targeted gene panels in 76 probands (64%) (2016-2021) or WGS in 43 (36%) (2021-2025). Clinical data were extracted to classify cases as syndromic or nonsyndromic and assess diagnostic outcomes, with complex cases discussed at MDTMs. RESULTS:Nonsyndromic (isolated or wider ocular) and syndromic cataracts accounted for 52.1% and 47.8% of cases, respectively, consistent with previous literature describing frequent multisystem involvement. WGS increased diagnostic yield by 10% compared with gene panels, primarily through detection of variants missed by panel-based approaches. Overall molecular diagnosis was 42%, with higher rates achieved in nonsyndromic forms (51.6%). CONCLUSION:WGS appears more effective for establishing a diagnosis in congenital cataracts than gene panels, because of its ability to detect diverse pathogenic variants. This is critical for provision of prognosis, reproductive counseling, and further management, especially given the high rate of associated syndromic conditions. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Our understanding of the genetic landscape of inherited optic neuropathies has grown significantly over the past decades, and it is now known to involve many genes found in both the nuclear and mitochondrial genomes, exhibiting all possible inheritance patterns. Furthermore, pathogenic variants in nuclear genes of mitochondrial respiratory Complex I (CI) subunits have been identified in some cases of ION, in addition to the more common severe presentation of CI deficiencies, usually with early onset. We conducted NGS screening of CI genes to identify potential causative variants in patients with optic atrophy, also performing comprehensive clinical assessments, including neuroimaging studies (MRI) and neurological evaluations. Detailed molecular structure modeling was performed to better evaluate the damaging effects of both novel and previously reported variants in the relevant CI subunits. We identified and characterized candidate causative variants in 31 patients from 23 unrelated families, with biallelic or hemizygous variants in 11 different nuclear CI-related genes encoding polypeptides involved in the structure of CI, including 3 core subunits (NDUFS7, NDUFV1, NDUFV2), 4 accessory subunits (NDUFA1, NDUFA10, NDUFA12, NDUFB11), and 4 assembly factors (NDUFAF2, NDUFAF3, NDUFAF4, NDUFAF8). Notably, defects in core CI subunits in this cohort lead to isolated optic atrophy, while defects in accessory CI subunits and assembly factors resulted in a spectrum of phenotypes, from isolated to syndromic optic atrophy. For 12 cases, the subacute onset of vision loss enabled us to associate or confirm novel genes (NDUFS7, NDUFV1, NDUFAF2, NDUFAF4, NDUFAF8) with the autosomal recessive Leber Hereditary Optic Neuropathy (arLHON) phenotype. Moreover, in the NDUFS7 subunit a partial spatial segregation was noted for missense variants causing either Leigh syndrome or isolated optic atrophy, hinting at possible disease-specific molecular defects. Our case series broadens the genetic spectrum of inherited optic neuropathies, emphasizing the crucial role of nuclear CI genes in its pathogenesis. The arLHON phenotype emerges as linked to numerous nuclear CI genes for which an insidious onset of optic atrophy is also reported, and in some cases the same variant may underlie both phenotypes. Overall, we highlight the possibly so far underestimated prevalence of CI nuclear subunits in the molecular diagnosis of ION, prompting to include all CI-related genes in the standard diagnostic screening.

Inflammatory linear verrucous epidermal nevus should be genotyped to direct treatment and genetic counselingTo the Editor: Inflammatory linear verrucous epidermal nevus (ILVEN) is a clinical diagnosis based on persistent Blaschko-linear erythematous scaly and usually pruritic lesions.Sixteen patients with a clinical diagnosis of ILVEN as defined by published diagnostic criteria were recruited and consented under Research Ethics Committee approval for phenotypic, histological, and genotypic analysis.At the time of initiating this study in 2014 no genetic causes of ILVEN were known.Causative genetic variants since described are in GJA1, 1 ABCA12 2 , CARD14, 3 PMVK 4 , NSDHL, 4 HRAS 4 and KRT10. 4Disease mechanisms thus far include germline X-linked variants, mosaic variants, and germline first hit with mosaic second hit.Paired blood and affected skin DNA underwent deep whole exome sequencing (WES, mean 250X), n ¼ 14, and if negative, skin DNA underwent targeted sequencing panel R327 (mosaic disorders, UK National Genomic Test Directory), n ¼ 8. Two patients had a negative WES and did not go forward to next generation sequencing panel due to sample limitations.Two patients recruited late in the study had next generation sequencing panel first and did not proceed to WES.We confirm here that ILVEN has multiple monogenic causes, with mutations in NSDHL (n ¼ 2, germline, NSDHL c.613G [ T, p.[G205T ], c.603_604delTG, p.[H201fs*69], both picked up on WES), PMVK (n ¼ 1, mosaic in blood and skin, no second variant detected in the same gene in skin, PMVK c.126delG, p.R42fs, picked up on WES), HRAS (n ¼ 1, mosaic, HRAS c.37G [ C, p.(G13R), picked up on panel, and CARD14 (n ¼ 2, mosaic, these 2 only previously published, 1 both picked up on WES).Ten patients had no pathogenic variants identified and we specifically excluded any variants in all previously described genes.No patients who were negative on WES had genes identified on a subsequent panel, suggesting that variants still unidentified are not in known mosaic genes, or if they are they are unlikely to be

Mosaic mutations in genes GNAQ or GNA11 lead to a spectrum of diseases including Sturge-Weber syndrome and phakomatosis pigmentovascularis with dermal melanocytosis. The pathognomonic finding of localized "tramlining" on plain skull radiography, representing medium-sized neurovascular calcification and associated with postnatal neurological deterioration, led us to study calcium metabolism in a cohort of 42 children. In this study, we find that 74% of patients had at least one abnormal measurement of calcium metabolism, the commonest being moderately low serum ionized calcium (41%) or high parathyroid hormone (17%). Lower levels of ionized calcium even within the normal range were significantly associated with seizures, and with specific antiepileptics despite normal vitamin D levels. Successive measurements documented substantial intrapersonal fluctuation in indices over time, and DEXA scans were normal in patients with hypocalcemia. Neurohistology from epilepsy surgery in five patients revealed not only intravascular, but perivascular and intraparenchymal mineral deposition and intraparenchymal microvascular disease in addition to previously reported findings. Neuroradiology review clearly demonstrated progressive calcium deposition in individuals over time. These findings and those of the adjoining paper suggest that calcium deposition in the brain of patients with GNAQ/GNA11 mosaicism may not be a nonspecific sign of damage as was previously thought, but may instead reflect the central postnatal pathological process in this disease spectrum.

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.

ObjectiveWe hypothesized that novel investigative pathways are needed to decrease diagnostic odysseys in pediatric mitochondrial disease and sought to determine the utility of clinical exome sequencing in a large cohort with suspected mitochondrial disease and to explore whether any of the traditional indicators of mitochondrial disease predict a confirmed genetic diagnosis.MethodsWe investigated a cohort of 85 pediatric patients using clinical exome sequencing and compared the results with the outcome of traditional diagnostic tests, including biochemical testing of routine parameters (lactate, alanine, and proline), neuroimaging, and muscle biopsy with histology and respiratory chain enzyme activity studies.ResultsWe established a genetic diagnosis in 36.5% of the cohort and report 20 novel disease-causing variants (1 mitochondrial DNA). Counterintuitively, routine biochemical markers were more predictive of mitochondrial disease than more invasive and elaborate muscle studies.ConclusionsWe propose using biochemical markers to support the clinical suspicion of mitochondrial disease and then apply first-line clinical exome sequencing to identify a definite diagnosis. Muscle biopsy studies should only be used in clinically urgent situations or to confirm an inconclusive genetic result.Classification of EvidenceThis is a Class II diagnostic accuracy study showing that the combination of CSF and plasma biochemical tests plus neuroimaging could predict the presence or absence of exome sequencing confirmed mitochondrial disorders.

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.

Background Primary 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. Methods The diagnostic utility of multigene panel next-generation sequencing (NGS) was evaluated in 161 unrelated families from multiple population ancestries. Results Most (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. Conclusions This 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.

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.

The authors developed a large next-generation DNA sequencing panel assay to screen for coding sequence and copy number variants in genes known to cause developmental eye disorders and inherited eye disease (429 genes); Sanger sequencing confirmed variants.1 They evaluated 277 probands, some of whom had multisystem disorders, using their panel and analyses. Of note, the overall diagnostic yield was relatively low at 68 of 277 samples (<25%).

AbstractBackgroundMitochondrial 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.MethodsTwo 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.ResultsIn 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.ConclusionsWe 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.

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.

IMPORTANCE Neuroinflammatory disorders are a range of severe neurological disorders causing brain and spinal inflammation and are now increasingly recognized in the pediatric population. They are often characterized by marked genotypic and phenotypic heterogeneity, complicating diagnostic work in clinical practice and molecular diagnosis. OBJECTIVE To develop and evaluate a next-generation sequencing panel targeting genes causing neuroinflammation or mimicking neuroinflammation. DESIGN, SETTING, AND PARTICIPANTS Cohort study in which a total of 257 genes associated with monogenic neuroinflammation and/or cerebral vasculopathy, including monogenic noninflammatory diseases mimicking these entities, were selected. A customized enrichment capture array, the neuroinflammation gene panel (NIP), was created. Targeted high-coverage sequencing was applied to DNA samples taken from eligible patients referred to Great Ormond Street Hospital in London, United Kingdom, between January 1, 2017, and January 30, 2019, because of onset of disease early in life, family history, and/or complex neuroinflammatory phenotypes. MAIN OUTCOMES AND MEASURES The main outcome was the percentage of individuals with definitive molecular diagnoses, variant classification, and clinical phenotyping of patients with pathogenic variants identified using the NIP panel. The NIP panel was initially validated in 16 patients with known genetic diagnoses. RESULTS The NIP was both sensitive (95%) and specific (100%) for detection of known mutations, including gene deletions, copy number variants, small insertions and deletions, and somatic mosaicism with allele fraction as low as 3%. Prospective testing of 60 patients (30 [50%] male; median [range] age, 9.8 [0.8-20] years) presenting with heterogeneous neuroinflammatory phenotypes revealed at least 1 class 5 (clearly pathogenic) variant in 9 of 60 patients (15%); 18 of 60 patients (30%) had at least 1 class 4 (likely pathogenic) variant. Overall, a definitive molecular diagnosis was established in 12 of 60 patients (20%). CONCLUSIONS AND RELEVANCE The NIP was associated with molecular diagnosis in this cohort and complemented routine laboratory and radiological workup of patients with neuroinflammation. Unexpected genotype-phenotype associations in patients with pathogenic variants deviating from the classic phenotype were identified. Obtaining an accurate molecular diagnosis in a timely fashion informed patient management, including successful targeted treatment in some instances and early institution of hematopoietic stem cell transplantation in others.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder of motile cilia dysfunction generally inherited as an autosomal recessive disease. Genetic testing is increasingly considered an early step in the PCD diagnostic workflow. We used targeted panel next-generation sequencing (NGS) for genetic screening of 33 Egyptian families with clinically highly suspected PCD. All variants prioritized were Sanger confirmed in the affected individuals and correctly segregated within the family. Targeted NGS yielded a high diagnostic output (70%) with biallelic mutations identified in known PCD genes. Mutations were identified in 13 genes overall, with CCDC40 and CCDC39 the most frequently mutated genes among Egyptian patients. Most identified mutations were predicted null effect variants (79%) and not reported before (85%). This study reveals that the genetic landscape of PCD among Egyptians is highly heterogeneous, indicating that a targeted NGS approach covering multiple genes will provide a superior diagnostic yield compared to Sanger sequencing for genetic diagnosis. The high diagnostic output achieved here highlights the potential of placing genetic testing early within the diagnostic workflow for PCD, in particular in developing countries where other diagnostic tests can be less available.