Detailed Analysis of ITPR1 Missense Variants Guides Diagnostics and Therapeutic Design.

BACKGROUND: The ITPR1 gene encodes the inositol 1,4,5-trisphosphate (IP3 ) receptor type 1 (IP3 R1), a critical player in cerebellar intracellular calcium signaling. Pathogenic missense variants in ITPR1 cause congenital spinocerebellar ataxia type 29 (SCA29), Gillespie syndrome (GLSP), and severe pontine/cerebellar hypoplasia. The pathophysiological basis of the different phenotypes is poorly understood. OBJECTIVES: We aimed to identify novel SCA29 and GLSP cases to define core phenotypes, describe the spectrum of missense variation across ITPR1, standardize the ITPR1 variant nomenclature, and investigate disease progression in relation to cerebellar atrophy. METHODS: Cases were identified using next-generation sequencing through the Deciphering Developmental Disorders study, the 100,000 Genomes project, and clinical collaborations. ITPR1 alternative splicing in the human cerebellum was investigated by quantitative polymerase chain reaction. RESULTS: We report the largest, multinational case series of 46 patients with 28 unique ITPR1 missense variants. Variants clustered in functional domains of the protein, especially in the N-terminal IP3 -binding domain, the carbonic anhydrase 8 (CA8)-binding region, and the C-terminal transmembrane channel domain. Variants outside these domains were of questionable clinical significance. Standardized transcript annotation, based on our ITPR1 transcript expression data, greatly facilitated analysis. Genotype-phenotype associations were highly variable. Importantly, while cerebellar atrophy was common, cerebellar volume loss did not correlate with symptom progression. CONCLUSIONS: This dataset represents the largest cohort of patients with ITPR1 missense variants, expanding the clinical spectrum of SCA29 and GLSP. Standardized transcript annotation is essential for future reporting. Our findings will aid in diagnostic interpretation in the clinic and guide selection of variants for preclinical studies. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Targeted next generation sequencing and family survey enable correct genetic diagnosis in CRX associated macular dystrophy - a case report.

BACKGROUND: We present 3 members of a family with macular dystrophy, originally diagnosed as Stargardt disease, with a significantly variable age at onset, caused by a heterozygous mutation in CRX. CASE PRESENTATION: A 43-year-old female with bull's eye maculopathy, whose sister was diagnosed with Stargardt disease previously at another centre, was found to have a single ABCA4 variant. Further examination of the family revealed that the asymptomatic father was also affected, indicating a dominant pattern of inheritance. In addition, the ABCA4 variant was not identified in the sister originally diagnosed with Stargardt disease. Next generation sequencing identified a heterozygous c.121C > T, p.R41W missense mutation in CRX in all 3 affected members. CONCLUSIONS: We describe a common phenotype, but with variable age at onset, with autosomal dominant inheritance and reduced penetrance in a family found to have a pathogenic sequence variant in CRX. This illustrates the importance of panel based molecular genetic testing accompanied by family studies to establish a definitive diagnosis.

"Genetic and clinical findings in an ethnically diverse retinitis pigmentosa cohort associated with pathogenic variants in EYS".

BACKGROUND AND OBJECTIVES: The EYS gene is an important cause of autosomal recessive retinitis pigmentosa (arRP). The objective of this study is to report on novel pathogenic variants in EYS and the range of associated phenotypes. SUBJECTS AND METHODS: This retrospective case series at a tertiary referral centre for inherited retinal diseases describes patients with an IRD and at least two variants in the EYS gene. Phenotyping included multimodal retinal imaging; genotyping molecular genetic analysis using targeted next generation sequencing. Sanger sequencing verification and analysis of novel variants using in silico approaches to determine their predicted pathogenicity. RESULTS: Eight male and four female patients were included. Age at onset ranged from 11 to 62 years with variable symptom presentation; ten patients showed classical features of retinitis pigmentosa, albeit with great variation in disease severity and extent. Two patients had atypical phenotypes: one with localised inferior sector pigmentation and a mild RP phenotype with changes predominantly at the posterior pole. Eighteen variants in EYS were identified, located across the gene: six were novel. Eight variants were missense, two altered splicing, one was a whole exon duplication and the remainder were predicted to result in premature truncation of the protein. CONCLUSION: The marked variability in severity and age of onset in most patients in this ethnically diverse cohort adds to growing evidence that that mild phenotypes are associated with EYS variants. Similarly, the two atypical cases add to the growing diversity of EYS disease as do the six novel pathogenic variants described.

Clinical Characterization of Retinitis Pigmentosa Associated With Variants in SNRNP200.

IMPORTANCE: SNRNP200 is a recently identified genetic cause of autosomal dominant retinitis pigmentosa (RP). However, the associated retinal phenotype is not well characterized. OBJECTIVE: To describe the retinal phenotype in patients with RP secondary to variants in SNRNP200. DESIGN, SETTING, AND PARTICIPANTS: This retrospective, case-series study was performed at 2 tertiary referral centers for inherited retinal diseases. Participants included 9 consecutive patients from 8 families with RP attributed to variants in SNRNP200. Data were collected from August 2017 to March 2018 and analyzed from May to July 2018. MAIN OUTCOMES AND MEASURES: Results of clinical evaluation, multimodal retinal imaging, and molecular genetic testing using targeted next-generation sequencing. RESULTS: Of the 9 patients included in the analysis (4 female and 5 male; mean [SD] age at presentation, 19 [15] years), each presented with nyctalopia, typically in the first 2 decades of life, although 2 patients experienced symptom onset in middle age. None had any consistent systemic features suggestive of syndromic RP. Retinal imaging studies and electroretinography findings were typical of a rod-predominant dystrophy with later involvement of cone photoreceptors. Phenotypic heterogeneity was typified by 4 unrelated patients with the common c.2041C>T SNRNP200 variant who demonstrated a variable age of disease onset (middle teenage years to the fourth decade of life). Disease progression was slow, with all but 1 patient maintaining visual acuity of better than 20/40 in the better-seeing eye in the fifth and sixth decades of life. CONCLUSIONS AND RELEVANCE: These data suggest that variants in SNRNP200 result in nonsyndromic RP with a typical phenotype of a rod-predominant dystrophy. Significant phenotypic heterogeneity and nonpenetrance were noted within some affected families. Symptom onset was typically within the first 2 decades of life, with slow progression and well-preserved visual acuities into the fifth and sixth decades.

Clinical and Molecular Characterization of PROM1-Related Retinal Degeneration.

Importance: The PROM1 gene, commonly associated with cone-rod dystrophies, may have dominant or recessive phenotypes that influence disease onset and severity. Objective: To characterize the clinical phenotype and molecular genetic variations in patients with PROM1 variants. Design, Setting, and Participants: This case-series study was conducted at 2 specialist retinal genetics clinics and examined 19 consecutively enrolled patients with PROM1-related retinal degeneration. Data were collected and analyzed from May 2018 to December 2018. Main Outcomes and Measures: Results of ophthalmic examination, retinal imaging, and molecular genetic analysis by next-generation sequencing. Results: Of 19 patients, 13 (68%) were women, and age ranged from 11 to 70 years. All patients presented with central visual loss, with or without photophobia. Individuals with recessive variants commonly had severe loss of visual acuity by their 20s, whereas the dominant variant was associated with a milder phenotype, with most patients retaining good vision into late adulthood. The recessive cases were associated with a panretinal dystrophy of cone-rod phenotype with early macular involvement, whereas the dominant variants were associated with a cone-rod phenotype that was restricted to the macula with predominantly cone dysfunction. Next-generation sequencing identified 3 novel and 9 previously reported variants in PROM1. Recessive mutations included 6 truncating variants (3 nonsense and 3 frameshift), 4 splice site variants, and 1 missense variant. All 6 dominant variants were associated with a c.1117C>T missense variant. The variants were distributed throughout the PROM1 genomic sequence with no specific clustering on protein domains. Conclusions and Relevance: In this case-series study, PROM1 recessive variants were associated with early-onset, severe panretinal degeneration. The similar phenotypes observed in patients with homozygous missense variants and splice site variants compared with similarly aged patients with truncating variants suggests that all recessive variants have a null (or loss of function close to null) outcome on PROM1 function. In contrast, the dominant missense cases were associated with a milder, cone-driven phenotype, suggesting that the dominant disease is preferentially associated with cones. This has implications for the development of treatments for this severely blinding disease, and adeno-associated viral vector-based gene therapy and optogenetics could become successful treatment options.

The Location of Exon 4 Mutations in RP1 Raises Challenges for Genetic Counseling and Gene Therapy.

PURPOSE: Mutations in the photoreceptor gene RP1 lead to recessive or dominantly inherited retinitis pigmentosa (RP). Since the dominantly inherited phenotype is generally milder than recessive cases, it raises the possibility that it could arise by haploinsufficiency; however, most mutations are in the terminal exon 4, which would be predicted to generate truncated proteins. We therefore assessed a cohort of RP patients with confirmed mutations in RP1 to examine the genetic basis of the exon 4 mutations. DESIGN: Observational case series. METHODS: A retrospective review of 15 patients, aged between 36 and 84, with RP1 mutations in exon 4 confirmed by Sanger sequencing. All patients underwent full ophthalmic examination. RESULTS: Two patients had homozygous mutations in RP1, p.(Glu1526*) and p.(Ser486fs), and presented with severe early-onset retinal degeneration. Their first-degree relatives were unaffected. Thirteen patients had dominantly inherited RP presenting in adult life with a rod-cone dystrophy phenotype. Four novel mutations were identified. All mutations were predicted to produce truncated RP1 protein of variable lengths, as follows: p.(Arg677*), p.(Gln679*), p.(Leu722*), p.(Ile725Argfs*6), p.(Ser734*)x2, p.(Leu762Tyrfs*17)x2, p.(Leu866Lysfs*7)x2, p.(Arg872Thrfs*2)x2, and p.(Gln917*). CONCLUSION: The RP1 protein with a predicted length between 677 and 917 amino acids seems to have a dominant negative effect, whereas proteins shorter (486 amino acids) or longer than this (1526 amino acids) lead to a more severe phenotype, but only in homozygous individuals. Since mutations at various points along exon 4 have divergent consequences, genetic testing alone may be insufficient for counseling, but recessive inheritance should be considered likely in severe early-onset cases.

Two Novel CAPN5 Variants Associated with Mild and Severe Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy Phenotypes.

Purpose: We report two new CAPN5 mutations associated with a phenotype of Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy. Methods: We performed next generation sequencing in two patients with ADNIV phenotype; the variants identified were explored further. Results: Patient 1 was heterozygous for CAPN5 c.799G>A, p.(Gly267Ser). Patient 2 was heterozygous for CAPN5 c.1126G>A, p.(Gly376Ser). Both amino acids are highly conserved across species. Patient 1 had a severe phenotype and his mutation lies within the protein's catalytic domain. Patient 2 had a mild phenotype and her mutation is the first ADNIV-causing mutation to be described in the regulatory domain of Calpain-5. Conclusions: Our findings potentially add two new ADNIV-causing CAPN5 mutations to the three previously described. We recommend CAPN5 genetic testing in all patients with a possible ADNIV phenotype, to develop our understanding of Calpain-5; a protein which could potentially provide therapeutically accessible targets for the treatment of many leading causes of blindness.

A splice-site variant in FLVCR1 produces retinitis pigmentosa without posterior column ataxia.

FLVCR1 (feline leukemia virus subgroup c receptor 1) is a transmembrane protein involved in the trafficking of intracellular heme. Homozygous variants in FLVCR1 have been described in association with a clinical syndrome of posterior column ataxia with retinitis pigmentosa (PCARP). Here, we describe a patient with non-syndromic retinitis pigmentosa homozygous for a splice-site variant in FLVCR1 (c.1092 + 5G>A) without evidence of posterior column ataxia or cerebellar degeneration. We suggest an association between intronic splice-site variants in FLVCR1 and the absence of posterior column degeneration and suggest a hypothesis to explain this observation. Should this association be proven, it would provide valuable prognostic information for patients. Retinal degeneration appears to be the sole clinical manifestation of this FLVCR1 variant; gene therapy approaches using an adeno-associated viral vector with sub-retinal delivery may therefore represent a therapeutic approach to halting retinal degeneration in this patient group.

Getting rhythm: how do babies do it?

OBJECTIVES: To investigate the emergence of biological rhythms in the first months of life in human infants, by measuring age-related changes in core body temperature during night-time sleep, hormones (cortisol and 6-sulfatoxymelatonin) and the expression of a clock-controlled gene H3f3b in oral epithelial cells. DESIGN: Observational longitudinal study. SETTING: We measured overnight core body temperature, actigraphy, day-night urinary cortisol and 6-sulfatoxymelatonin, as well as circadian gene expression, in infants at home from March 2007 to July 2008 in Leicester. PARTICIPANTS: We recruited 35 healthy Caucasian infants who were born at term. They were monitored from 6 to 18 weeks of age. RESULTS: At 8 weeks of age the day-night rhythm of cortisol secretion was the first to appear followed by 6-sulfatoxymelatonin 1 week later; at the same time that night-time sleep was established. At 10 weeks, the maximum fall in deep body temperature occurred with the onset of night-time sleep, followed at 11 weeks by the rhythmical expression of the H3f3b gene. CONCLUSIONS: In human infants, there is a clear sequential pattern for the emergence of diurnal biological rhythms between 6 and 18 weeks of postnatal age, led by the secretion of cortisol and linked with the establishment of consolidated night-time sleep. It is likely that this represents part of a maturation and adaption process as infants gain equilibrium with their external environment after birth.

Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model.

Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the 'diagnostic odyssey' for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1-3, 6, 7 and Friedrich's ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3-35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US$620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.

Next-generation sequencing (NGS) as a diagnostic tool for retinal degeneration reveals a much higher detection rate in early-onset disease.

Inherited retinal degeneration (IRD) is a common cause of visual impairment (prevalence ∼1/3500). There is considerable phenotype and genotype heterogeneity, making a specific diagnosis very difficult without molecular testing. We investigated targeted capture combined with next-generation sequencing using Nimblegen 12plex arrays and the Roche 454 sequencing platform to explore its potential for clinical diagnostics in two common types of IRD, retinitis pigmentosa and cone-rod dystrophy. 50 patients (36 unknowns and 14 positive controls) were screened, and pathogenic mutations were identified in 25% of patients in the unknown, with 53% in the early-onset cases. All patients with new mutations detected had an age of onset <21 years and 44% had a family history. Thirty-one percent of mutations detected were novel. A de novo mutation in rhodopsin was identified in one early-onset case without a family history. Bioinformatic pipelines were developed to identify likely pathogenic mutations and stringent criteria were used for assignment of pathogenicity. Analysis of sequencing metrics revealed significant variability in capture efficiency and depth of coverage. We conclude that targeted capture and next-generation sequencing are likely to be very useful in a diagnostic setting, but patients with earlier onset of disease are more likely to benefit from using this strategy. The mutation-detection rate suggests that many patients are likely to have mutations in novel genes.

Next-generation sequencing in health-care delivery: lessons from the functional analysis of rhodopsin.

PURPOSE: The interpretation of genetic information has always been challenging, but next-generation sequencing produces data on such a vast scale that many more variants of uncertain pathogenicity will be found. We exemplify this issue with reference to human rhodopsin, in which pathogenic mutations can lead to autosomal dominant retinitis pigmentosa. METHODS: Rhodopsin variants, with unknown pathogenicity, were found in patients by next-generation and Sanger sequencing and a multidisciplinary approach was used to determine their functional significance. RESULTS: Four variants in rhodopsin were identified: F45L, P53R, R69H, and M39R, with the latter two substitutions being novel. We investigated the cellular transport and photopigment function of all four human substitutions and found that the F45L and R69H variants behave like wild-type and are highly unlikely to be pathogenic. By contrast, P53R (a de novo change) and M39R were retained in the endoplasmic reticulum with significantly reduced functionality and are clearly pathogenic. CONCLUSION: Potential pathogenicity of variants requires careful assessment using clinical, genetic, and functional data. We suggest that a multidisciplinary pathway of assessment, using several functional assays, will be required if next-generation sequencing is to be used effectively, reliably, and safely in the clinical environment.

The effects of in utero irradiation on mutation induction and transgenerational instability in mice.

Epidemiological evidence suggests that the deleterious effects of prenatal irradiation can manifest during childhood, resulting in an increased risk of leukaemia and solid cancers after birth. However, the mechanisms underlying the long-term effects of foetal irradiation remain poorly understood. This study was designed to analyse the impact of in utero irradiation on mutation rates at expanded simple tandem repeat (ESTR) DNA loci in directly exposed mice and their first-generation (F(1)) offspring. ESTR mutation frequencies in the germline and somatic tissues of male and female mice irradiated at 12 days of gestation remained highly elevated during adulthood, which was mainly attributed to a significant increase in the frequency of singleton mutations. The prevalence of singleton mutations in directly exposed mice suggests that foetal irradiation results in genomic instability manifested both in utero and during adulthood. The frequency of ESTR mutation in the F(1) offspring of prenatally irradiated male mice was equally elevated across all tissues, which suggests that foetal exposure results in transgenerational genomic instability. In contrast, maternal in utero exposure did not affect the F(1) stability. Our data imply that the passive erasure of epigenetic marks in the maternal genome can diminish the transgenerational effects of foetal irradiation and therefore provide important clues to the still unknown mechanisms of radiation-induced genomic instability. The results of this study offer a plausible explanation for the effects of in utero irradiation on the risk of leukaemia and solid cancers after birth.

Complex germline and somatic mutation processes at a haploid human minisatellite shown by single-molecule analysis.

Mutation at most human minisatellites is driven by complex interallelic processes that give rise to a high degree of length polymorphism and internal structural variation. MSY1, the only highly variable minisatellite on the non-recombining region of the Y chromosome, is constitutively haploid and therefore precluded from interallelic interactions, yet maintains high diversity in both length and structure. To investigate the basis of its mutation processes, an unbiased structural analysis of >500 single-molecule MSY1 PCR products from matched sperm and blood samples from a single donor was undertaken. The overall mutation frequencies in sperm and blood DNAs were not significantly different, at 2.68% and 1.88%, respectively. Sperm DNA showed significantly more length mutants than blood DNA, with mutants in both tissues involving small-scale (1-3 repeat units in a 77 repeat progenitor allele) increases or decreases in repeat block lengths, with no gain or loss bias. Isometric mutations altering structure but not length were found in both tissues, and involved either the apparent shift of a boundary between repeat unit blocks (a 'boundary switch') or the conversion of a repeat within a block to a different repeat type ('modular structure' mutant). There was a significant excess of boundary switch mutants and deficit of modular structure mutants in sperm. A comparison of mutant structures with phylogenetically matched alleles in population samples showed that alleles with structures resembling the blood mutants were unlikely to arise in populations. Mutation seems likely to involve gene conversion via synthesis-dependent strand annealing, and the blood-sperm differences may reflect more relaxed constraint on sister chromatid alignment in blood.

Thomas Jefferson's Y chromosome belongs to a rare European lineage.

We have characterized the Y chromosome carried by President Thomas Jefferson, the general rarity of which supported the idea that he, or a patrilineal relative, fathered the last son of his slave Sally Hemings. It belongs to haplogroup K2, a lineage representing only approximately 1% of chromosomes worldwide, and most common in East Africa and the Middle East. Phylogenetic network analysis of its Y-STR (short tandem repeat) haplotype shows that it is most closely related to an Egyptian K2 haplotype, but the presence of scattered and diverse European haplotypes within the network is nonetheless consistent with Jefferson's patrilineage belonging to an ancient and rare indigenous European type. This is supported by the observation that two of 85 unrelated British men sharing the surname Jefferson also share the President's Y-STR haplotype within haplogroup K2. Our findings represent a cautionary tale in showing the difficulty of assigning individual ancestry based on a Y-chromosome haplotype, particularly for rare lineages where population data are scarce.