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.

Inherited retinal degeneration (IRD) is a common cause of visual impairment (prevalence B1/3500). 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 o21 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.

It is well known that the developing embryo is especially sensitive to ionising radiation. However, to date little is known about the long-term effects of in utero exposure on mutation rates during adulthood. To evaluate the effects of in utero irradiation on mutation induction and transgenerational instability, BALB/c pregnant mice (Theiler stage 20, 12 days of gestation) were exposed to 1 Gy of acute X-rays. The in utero exposed 8-week-old males and females were mated to control partners. To evaluate the effects of in utero irradiation on mutation induction in the germline of exposed mice, all parents and offspring were profiled using two mouse-specific expanded simple tandem repeat (ESTR) probes Ms6-hm and Hm-2. The results of our study show that ESTR mutation rates in the germline of in utero irradiated male and female mice remain highly elevated during adulthood. Using single-molecule PCR, the frequency of ESTR mutation was established in DNA samples prepared from sperm, bone marrow and brain taken from the in utero irradiated animals. In all animals, a statistically significant ∼2.8-3.7 fold increase in the mean mutation frequency was found in all tissues of the in utero irradiated animals. The results of our study show that the mutagenic effects of in utero irradiation in mice are well manifested during adulthood and therefore suggest that the susceptibility of early stages of mouse development to ionising radiation may be higher than previously thought. To analyse the effects of parental irradiation on transgenerational instability, the frequency of ESTR mutation was established in DNA samples prepared from sperm, bone marrow and brain taken from the first-generation offspring of in utero irradiated male and female mice. The results of our study show that in the offspring of in utero exposed males the frequency of ESTR mutation is considerably elevated across multiple tissues, whereas in the offspring of irradiated females it does not significantly differ from that in controls. A comparison with the results of our previous studies on transgenerational in stability among the offspring of BALB/c male mice irradiated during adulthood showed that that the magnitude of transgenerational effects is not affected by the stage of paternal exposure. This work has therefore established that an instability signal induced in the germline of in utero irradiated males is manifested during adulthood. The potential implications of our findings to for further understanding of the possible mechanisms of transgenerational genomic instability will be discussed.

Key Points Question What are the clinical and molecular characteristics of PROM1- related retinal degeneration? Findings In this case series of 19 patients with PROM1 -related retinal degeneration, recessive variants were associated with early-onset, severe panretinal degeneration, whereas the dominant disease was associated with the c.1117C>T variant and a late-onset, milder phenotype that predominantly involves the macula. In addition, the dominant variant was preferentially associated with cone photoreceptors. Meaning A better understanding of the clinical and molecular characteristics of PROM1- related retinal degeneration may aid development of future treatments, including gene therapy and optogenetics.

original research articlePurpose: 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. 2012:14(11):891-899 Genet Med

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It is well known that the developing embryo is especially sensitive to ionising radiation. However, to date little is known about the long-term effects of in utero exposure on mutation rates during adulthood. To evaluate the effects of in utero irradiation on mutation induction and transgenerational instability, BALB/c pregnant mice (Theiler stage 20, 12 days of gestation) were exposed to 1 Gy of acute X-rays. The in utero exposed 8-week-old males and females were mated to control partners. To evaluate the effects of in utero irradiation on mutation induction in the germline of exposed mice, all parents and offspring were profiled using two mouse-specific expanded simple tandem repeat (ESTR) probes Ms6-hm and Hm-2. The results of our study show that ESTR mutation rates in the germline of in utero irradiated male and female mice remain highly elevated during adulthood. Using single-molecule PCR, the frequency of ESTR mutation was established in DNA samples prepared from sperm, bone marrow and brain taken from the in utero irradiated animals. In all animals, a statistically significant ∼2.8-3.7 fold increase in the mean mutation frequency was found in all tissues of the in utero irradiated animals. The results of our study show that the mutagenic effects of in utero irradiation in mice are well manifested during adulthood and therefore suggest that the susceptibility of early stages of mouse development to ionising radiation may be higher than previously thought. To analyse the effects of parental irradiation on transgenerational instability, the frequency of ESTR mutation was established in DNA samples prepared from sperm, bone marrow and brain taken from the first-generation offspring of in utero irradiated male and female mice. The results of our study show that in the offspring of in utero exposed males the frequency of ESTR mutation is considerably elevated across multiple tissues, whereas in the offspring of irradiated females it does not significantly differ from that in controls. A comparison with the results of our previous studies on transgenerational in stability among the offspring of BALB/c male mice irradiated during adulthood showed that that the magnitude of transgenerational effects is not affected by the stage of paternal exposure. This work has therefore established that an instability signal induced in the germline of in utero irradiated males is manifested during adulthood. The potential implications of our findings to for further understanding of the possible mechanisms of transgenerational genomic instability will be discussed.

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.