The impact of inversions across 33,924 families with rare disease from a national genome sequencing project.

Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.

Evaluation of tumour surveillance protocols and outcomes in von Hippel-Lindau disease in a national health service.

BACKGROUND: Von Hippel-Lindau (VHL) disease is an inherited tumour predisposition syndrome and a paradigm for the importance of early diagnosis and surveillance. However, there is limited information on the "real world" management of VHL disease. METHODS: A national audit of VHL disease in the United Kingdom. RESULTS: VHL disease was managed mostly via specialist clinics coordinated through regional clinical genetics services (but frequently involving additional specialties). Over the study period, 19 genetic centres saw 842 individuals (393 males, 449 females) with a clinical and/or molecular diagnosis of VHL disease and 74 individuals (35 male, 39 female) with a prior risk of 50% (affected parent). All centres offered retinal, central nervous system and abdominal surveillance to affected individuals and at-risk relatives though surveillance details differed between centres (but complied with international recommendations). Renal lesions detected on the first surveillance scan were, on average, larger than those detected during subsequent scans and the larger the diameter at detection the greater the likelihood of early intervention. CONCLUSIONS: In a state-funded health care system individuals with a rare inherited cancer predisposition syndrome are generally able to access appropriate surveillance and patient management is improved compared to historical data. The "real world" data from this study will inform the future development of VHL management protocols.

Expanding the FDXR-Associated Disease Phenotype: Retinal Dystrophy Is a Recurrent Ocular Feature.

Purpose: The purpose of this study was to report retinal dystrophy as a novel clinical feature and expand the ocular phenotype in patients harboring biallelic candidate FDXR variants. Methods: Patients carrying biallelic candidate FDXR variants were identified by whole genome sequencing (WGS) as part of the National Institute for Health Research BioResource rare-disease and the UK's 100,000 Genomes Project (100KGP) with an additional case identified by exome sequencing. Retrospective clinical data were collected from the medical records. Haplotype reconstruction was performed in families harboring the same missense variant. Results: Ten individuals from 8 unrelated families with biallelic candidate variants in FDXR were identified. In addition to bilateral optic atrophy and variable extra-ocular findings, 7 of 10 individuals manifested retinal dystrophy comprising dysfunction and degeneration of both rod and cone photoreceptors. Five of 10 subjects had sensorineural hearing loss. The previously unreported missense variant (c.1115C > A, p.(Pro372His)) was found in 5 of 8 (62.5%) study families. Haplotype reconstruction using WGS data demonstrated a likely ancestral haplotype. Conclusions: FDXR-associated disease is a phenotypically heterogeneous disorder with retinal dystrophy being a major clinical feature observed in this cohort. In addition, we hypothesize that a number of factors are likely to drive the pathogenesis of optic atrophy, retinal degeneration, and perhaps the associated systemic manifestations.

Ligase IV syndrome can present with microcephaly and radial ray anomalies similar to Fanconi anaemia plus fatal kidney malformations.

Ligase IV (LIG4) syndrome is a rare disorder of DNA damage repair caused by biallelic, pathogenic variants in LIG4. This is a phenotypically heterogeneous condition with clinical presentation varying from lymphoreticular malignancies in developmentally normal individuals to significant microcephaly, primordial dwarfism, radiation hypersensitivity, severe combined immunodeficiency and early mortality. Renal defects have only rarely been described as part of the ligase IV disease spectrum. We identified a consanguineous family where three siblings presenting with antenatal growth retardation, microcephaly, severe renal anomalies and skeletal abnormalities, including radial ray defects. Autozygosity mapping and exome sequencing identified a novel homozygous frameshift variant in LIG4, c.597_600delTCAG, p.(Gln200LysfsTer33), which segregated in the family. LIG4 is encoded by a single exon and so this frameshift variant is predicted to result in a protein truncated by 678 amino acids. This is the shortest predicted LIG4 protein product reported and correlates with the most severe clinical phenotype described to date. We note the clinical overlap with Fanconi anemia and suggest that LIG4 syndrome is considered in the differential diagnosis of this severe developmental disorder.

Cranial meningiomas in 411 neurofibromatosis type 2 (NF2) patients with proven gene mutations: clear positional effect of mutations, but absence of female severity effect on age at onset.

BACKGROUND: Meningiomas have been reported to occur in approximately 50% of neurofibromatosis type 2 (NF2) patients. The NF2 gene is commonly biallelically inactivated in both schwannomas and meningiomas. The spectrum of NF2 mutations consists mainly of truncating (nonsense and frameshift) mutations. A smaller number of patients have missense mutations, which are associated with a milder disease phenotype. METHODS: This study analysed the cumulative incidence and gender effects as well as the genotype-phenotype correlation between the position of the NF2 mutation and the occurrence of cranial meningiomas in a cohort of 411 NF2 patients with proven NF2 mutations. RESULTS AND CONCLUSION: Patients with mutations in exon 14 or 15 were least likely to develop meningiomas. Cumulative risk of cranial meningioma to age 50 years was 70% for exons 1-3, 81% for exons 4-6, 49% for exons 7-9, 56% for exons 10-13, and 28% for exons 14-15. In the cohort of 411 patients, no overall gender bias was found for occurrence of meningioma in NF2 disease. Cumulative incidence of meningioma was close to 80% by 70 years of age for both males and females, but incidence by age 20 years was slightly increased in males (male 25%, female 18%; p=0.023). Conversely, an increased risk of meningiomas in women with mosaic NF2 disease was also found.

Are patients with intermediate TPMT activity at increased risk of myelosuppression when taking thiopurine medications?

UNLABELLED: Thiopurine S-methyltransferase (TPMT) metabolizes thiopurine medications, including azathioprine and 6-mercaptopurine. Absent TPMT activity (i.e., in individuals homozygous for a variant TPMT allele) is associated with an increased risk of myelosuppression in patients taking thiopurine drugs. However, it is not clear if there is also an increased risk for patients with intermediate TPMT activity (i.e., in individuals heterozygous for a variant TPMT allele). AIMS: To quantify the increased risk of myelosuppression for patients with intermediate TPMT activity. MATERIALS & METHODS: A systematic review identified published studies, up to 29 September 2008, that explored the relationship between TMPT and hematological adverse drug reactions to thiopurines. Following a critical appraisal of the quality of published studies, a meta-analysis calculated the odds ratio of myelosuppression for patients with intermediate TPMT activity compared with wild-type. RESULTS: A total of 67 studies were identified, the majority retrospective cohort in design. Patients with two TPMT variant alleles who are TPMT deficient have a substantial increase in their risk of myelotoxicity (86% of deficient patients developed myelosuppression). The increase in odds ratio of developing leukopenia for patients with intermediate TPMT activity or one TPMT variant allele compared with wild-type was 4.19 (95% CI: 3.20-5.48). CONCLUSION: This meta-analysis suggests that individuals with both intermediate and absent TPMT activity have an increased risk of developing thiopurine-induced myelosuppression, compared with individuals with normal activity. However, there is significant variability in the quality of the reported studies and large prospective studies to clarify the size of the effect of TPMT variant alleles on the risk of myelosuppression should be conducted. Accurate risk assessments will provide important data to inform clinical guidelines.

Pharmacogenetic testing in the United Kingdom genetics and immunogenetics laboratories.

AIM: For certain drugs, pharmacogenetic tests can reduce adverse drug reactions and improve treatment efficacy. However, the adoption of pharmacogenetics into clinical practice has been relatively slow. One potential barrier is the capacity of laboratories to meet the demands of a clinical pharmacogenetic service. We aimed to establish the range, capacity to deliver, and demand for germline pharmacogenetic testing in the United Kingdom and Ireland, through an e-survey of 34 molecular genetics and 28 histocompatibility and immunogenetics (H&I) laboratories. RESULTS: Thirty-five percent of molecular genetics laboratories and 54% of H&I laboratories responded to the survey. The majority of H&I laboratories (93%) offered pharmacogenetic testing, whereas only one molecular genetics laboratory provided a pharmacogenetic service. HLA-B*5701 was most commonly tested to identify those at risk of abacavir hypersensitivity among patients with HIV. A number of barriers to testing were identified, including lack of clinician knowledge and a lack of scientific evidence. All molecular genetics laboratories believed that national coordination of clinical pharmacogenetic services was required, whereas only 50% of H&I laboratories supported this view. CONCLUSIONS: In the United Kingdom, pharmacogenetic testing is currently being predominantly provided through H&I laboratories for a limited number of indications. The number of laboratories offering pharmacogenetic tests is increasing and is likely to continue to increase over the coming years.

Pharmacogenetics education in British medical schools.

Pharmacogenetic tests allow medications to be tailored to individual patients to improve efficacy and reduce drug toxicity. In 2005, the International Society of Pharmacogenomics (ISP) made recommendations for undergraduate medical teaching in pharmacogenetics. We aimed to establish the quantity and scope of this in British medical schools. An electronic survey was sent to all British medical schools. Nineteen out of 34 (56%) medical schools responded. Sixteen of the 19 (84%) respondents provided pharmacogenetics teaching, usually 1-2 h in total. Only four (21%) medical schools offered the four or more hours of teaching recommended by the ISP. However, 10 of 16 (63%) schools felt the amount of pharmacogenetic teaching offered was sufficient. The quantity of undergraduate teaching of pharmacogenetics is low. However, a majority of UK medical schools teach it, covering a broad scope of elements. It is encouraging that future clinicians are being provided with the knowledge to deliver pharmacogenetics into clinical practice.

The first deltex null mutant indicates tissue-specific deltex-dependent Notch signaling in Drosophila.

Notch (N) is a single-pass transmembrane receptor. The N signaling pathway is an evolutionarily conserved mechanism that controls various cell-specification processes. Drosophila Deltex (Dx), a RING-domain E3 ubiquitin ligase, binds to the N intracellular domain, promotes N's endocytic trafficking to late endosomes, and was proposed to activate Suppressor of Hairless [Su(H)]-independent N signaling. However, it has been difficult to evaluate the importance of dx, because no null mutant of a dx family gene has been available in any organism. Here, we report the first null mutant allele of Drosophila dx. We found that dx was involved only in the subsets of N signaling, but was not essential for it in any developmental context. A strong genetic interaction between dx and Su(H) suggested that dx might function in Su(H)-dependent N signaling. Our epistatic analyses suggested that dx functions downstream of the ligands and upstream of activated Su(H). We also uncovered a novel dx activity that suppressed N signaling downstream of N.

Regulation of notch endosomal sorting and signaling by Drosophila Nedd4 family proteins.

The Notch receptor mediates a short-range signal that regulates many cell fate decisions. The misregulation of Notch has been linked to cancer and to developmental disorders. Upon binding to its ligands, Delta (Dl) or Serrate (Ser), the Notch ectodomain is shed by the action of an ADAM protease. The Notch intracellular domain is subsequently released proteolytically from the membrane by Presenilin and translocates to the nucleus to activate the transcription factor, Suppressor of Hairless. We show in Drosophila that Notch signaling is limited by the activity of two Nedd4 family HECT domain proteins, Suppressor of deltex [Su(dx)] and DNedd4. We rule out models by which Su(dx) downregulates Notch through modulating Deltex or by limiting the adherens junction accumulation of Notch. Instead, we show that Su(dx) regulates the postendocytic sorting of Notch within the early endosome to an Hrs- and ubiquitin-enriched subdomain en route to the late endosome. We propose a model in which endocytic sorting of Notch mediates a decision between its activation and downregulation. Such intersections between trafficking routes may provide key points at which other signals can modulate Notch activity in both normal development and in the pathological misactivation of Notch.

Mitochondrial profiling of dog hairs.

In order to make greater use of dog hairs as forensic evidence, we have developed a robust method for duplex amplification of adjacent 306 and 332bp amplicons within the 5' hypervariable region (5' HVR) of the canine mitochondrial control region. In support of this, a 595bp region covering 35 polymorphic sites has been sequenced from the blood of 105 UK dogs. In total, 30 different haplotypes were observed, 13 only once whilst the commonest was seen 14 times; the overall exclusion capacity is 0.929. One animal was heteroplasmic in blood for a single base deletion and showed phenotypes ranging from near complete deletion to a predominance of the base among a sample of 12 hairs. In contrast, no evidence of heteroplasmy was seen in single hairs from 20 dogs which were not visibly heteroplasmic in blood. Phylogenetic analysis and comparisons with other published databases highlighted instances of possible recurrent mutation which may be relevant when interpreting single base differences between samples.