Exonic mutations in cell-cell adhesion may contribute to CADASIL-related CSVD pathology.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a condition caused by mutations in NOTCH3 and results in a phenotype characterised by recurrent strokes, vascular dementia and migraines. Whilst a genetic basis for the disease is known, the molecular mechanisms underpinning the pathology of CADASIL are still yet to be determined. Studies conducted at the Genomics Research Centre (GRC) have also identified that only 15-23% of individuals clinically suspected of CADASIL have mutations in NOTCH3. Based on this, whole exome sequencing was used to identify novel genetic variants for CADASIL-like cerebral small-vessel disease (CSVD). Analysis of functionally important variants in 50 individuals was investigated using overrepresentation tests in Gene ontology software to identify biological processes that are potentially affected in this group of patients. Further investigation of the genes in these processes was completed using the TRAPD software to identify if there is an increased number (burden) of mutations that are associated with CADASIL-like pathology. Results from this study identified that cell-cell adhesion genes were positively overrepresented in the PANTHER GO-slim database. TRAPD burden testing identified n = 15 genes that had a higher number of rare (MAF < 0.001) and predicted functionally relevant (SIFT < 0.05, PolyPhen > 0.8) mutations compared to the gnomAD v2.1.1 exome control dataset. Furthermore, these results identified ARVCF, GPR17, PTPRS, and CELSR1 as novel candidate genes in CADASIL-related pathology. This study identified a novel process that may be playing a role in the vascular damage related to CADASIL-related CSVD and implicated n = 15 genes in playing a role in the disease.

GSTM1 and GSTT1 polymorphisms associated with pain in a chemotherapy-induced peripheral neuropathy cohort.

PURPOSE: Chemotherapy induced peripheral neuropathy (CIPN) is a debilitating condition that is a direct consequence of receiving cancer treatment. The molecular aetiology of CIPN is not well understood, and it is theorised that there may be a genetic component. Genetic polymorphisms in Glutathione-S Transferase (GST) genes, including GSTT1, GSTM1 and GSTP1, encode for enzymes known to metabolise drugs used in chemotherapy, and have been theorised to be associated with CIPN. This study aimed to investigate four markers in these genes for an association in a mixed cancer cohort in relation to CIPN (n = 172). METHODS: CIPN was measured using the neuropathy item from the Patient Reported Outcome Common Terminology Criteria for Adverse Event (PRO-CTCAE) assessment. Genotyping for all samples was performed using PCR for the GSTM1 and GSTT1 null variants and restriction fragment length polymorphisms for the GSTP1 and GSTM1 polymorphisms. RESULTS: No associations were found for the GST gene markers in relation to CIPN within our study, or CIPN severity. Longitudinal stratification of the CIPN phenotypes to examine links for neuropathy, identified nominally significant protective associations with the GSTM* null allele (p-value = 0.038, OR = 0.55) and the presence of pain at month 2 of treatment, as well as a risk factor for pain related month 2 of treatment for individuals with the GSTT1*null allele (p-value = 0.030, OR = 1.64). Higher severity of pain in patients with CIPN persisted at each time-point compared to those without CIPN. CONCLUSION: No significant results for an association between CIPN with polymorphisms in GSTM1, GSTT1 and GSTP1 were identified. However, associations for the GSTM1¬-null and GSTT1-null polymorphisms with pain at month 2 following chemotherapy were identified.

Investigating a Genetic Link Between Alzheimer's Disease and CADASIL-Related Cerebral Small Vessel Disease.

Monogenic forms of Alzheimer's disease (AD) have been identified through mutations in genes such as APP, PSEN1, and PSEN2, whilst other genetic markers such as the APOE ε carrier allele status have been shown to increase the likelihood of having the disease. Mutations in these genes are not limited to AD, as APP mutations can also cause an amyloid form of cerebral small vessel disease (CSVD) known as cerebral amyloid angiopathy, whilst PSEN1 and PSEN2 are involved in NOTCH3 signalling, a process known to be dysregulated in the monogenic CSVD, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). The overlap between AD genes and causes of CSVD led to the hypothesis that mutations in other genes within the PANTHER AD-presenilin pathway may be novel causes of CSVD in a cohort of clinically suspicious CADASIL patients without a pathogenic NOTCH3 mutation. To investigate this, whole exome sequencing was performed on 50 suspected CADASIL patients with no NOTCH3 mutations, and a targeted gene analysis was completed on the PANTHER. ERN1 was identified as a novel candidate CSVD gene following predicted pathogenic gene mutation analysis. Rare variant burden testing failed to identify an association with any gene; however, it did show a nominally significant link with ERN1 and TRPC3. This study provides evidence to support a genetic overlap between CSVD and Alzheimer's disease.

The novel allele HLA-DQA1*05:05:10 was detected in a Nigerian who also lacks the DRB5 gene in the DRB1*16 haplotype.

One synonymous nucleotide substitution in exon 2 of DQA1*05:05:01:13 results in a novel allele, HLA-DQA1*05:05:10.

Detection of a novel allele, HLA-DPA1*01:58, originating from a recombination within exon 2 of the DPA1 gene.

A crossover in exon 2 between DPA1*01:03:01:02 and DPA1*02:01:01:01 results in a novel hybrid allele, HLA-DPA1*01:58.

The MinION as a cost-effective technology for diagnostic screening of the SCN1A gene in epilepsy patients.

The MinION is a portable DNA sequencer that allows real time sequencing at low capital cost investment. We assessed accuracy and cost-effectivess of the MinION for genetic diagnostic testing of known SCN1A mutations that cause Dravet Syndrome (DS). DNA samples (n = 7) from DS patients previously shown to carry SCN1A mutations via Ion Torrent and Sanger sequencing were sequenced using the MinION. SCN1A amplicons for 8 exons were sequenced using the MinION with 1D chemistry on an R9.4 flow cell. All known missense mutations were detected in all samples showing 100 % concordance with results from other methods. However, the MinION failed to detect the insertions/deletions (INDELs) present in these patients. Nevertheless, these results indicate that MinION is a cost-effective platform for use as an initial screening step in the detection of nucleotide substitution mutations in in SCN1A, especially in under-resourced laboratories or hospitals. Further improvements are required to reliably detect INDELS in this gene.

BSHI guideline: HLA matching and donor selection for haematopoietic progenitor cell transplantation.

A review of the British Society for Histocompatibility and Immunogenetics (BSHI) Guideline 'HLA matching and donor selection for haematopoietic progenitor cell transplantation' published in 2016 was undertaken by a BSHI appointed writing committee. Literature searches were performed and the data extracted were presented as recommendations according to the GRADE nomenclature.

Tiered analysis of whole-exome sequencing for epilepsy diagnosis.

It is thought that despite highly variable phenotypic expression, 70-80% of all epileptic cases are caused by one or more genetic mutations. Next generation sequencing technologies, such as whole exome sequencing (WES), can be used in a diagnostic or research setting to identify genetic mutations which may have significant prognostic implications for patients and their families. In this study, 398 genes associated with epilepsy or recurrent seizures were stratified into tiers based on genotype-phenotype concordance, tissue gene expression, frequency of affected individuals with mutations and evidence from functional and family studies. WES was completed on 14 DNA samples (2 with known mutations in SCN1A and 12 with no known mutations) from individuals diagnosed with epilepsy using an Ion AmpliSeq approach. WES confirmed positive SCN1A mutations in two samples. In n = 5/12 samples (S-3 to -14) we identified potentially causative mutations across five different genes. S-5 was identified to have a novel missense mutation in CCM2; S-6 a novel frameshift mutation identified in ADGRV1; S-10 had a previously reported pathogenic mutation in PCDH19, whilst a novel missense mutation in PCDH19 was shown in S-12; and S-13 identified to have separate missense mutations in KCNA2 and NPRL3. The application of WES followed by a targeted variant prioritization approach allowed for the discovery of potentially causative mutations in our cohort of previously undiagnosed epilepsy patients.

Variant Call Format-Diagnostic Annotation and Reporting Tool: A Customizable Analysis Pipeline for Identification of Clinically Relevant Genetic Variants in Next-Generation Sequencing Data.

In this article, we introduce the variant call format-diagnostic annotation and reporting tool (VCF-DART), a customized analysis pipeline tool for the rapid annotation of variants from exome or genome sequencing and the generation of reports for genetic diagnostics. VCF-DART uses custom gene lists to categorize variants into specific analysis tiers and to subcategorize them on the basis of standard parameters to facilitate the rapid interrogation of potentially pathogenic variants by human operators. The tool uses publicly available databases to identify a range of data to assist with variant classification and curation processes and includes robust logging of parameters and database versions to allow comparison of analyses performed at different times. VCF-DART-an online analysis pipeline for next-generation sequencing data-is a platform agnostic tool that leverages the use of publicly available databases to improve a laboratory's calling ability and reduce analysis times long-term. It also runs highly efficiently and scales from desk and laptop machines to servers. Overall, VCF-DART provides a simple, customizable, and entirely open-source method to identify genetic variants that may be of clinical importance in a variety of genetically important conditions.

Does education attenuate the genetic risk of obesity? Evidence from U.K. Twins.

More education is associated with a lower body mass index (BMI) and likelihood of being obese. Obesity and BMI also have a strong genetic basis. Given these observations, we investigate whether more education can reduce obesity by attenuating the underlying genetic risk of being obese, through gene-environment (GxE) interactions. We estimate associations between (i) education, (ii) a genetic risk score (GRS) and (iii) GxE interactions between education and the GRS through Ordinary least Squares (OLS) and twins fixed-effect regressions using data on female twins from the TwinsUK database. OLS estimates show that there are significant associations of education and genetics. Female twins with a university education are 14.3 percentage points less likely to be obese compared to twins with less than compulsory education, and a 1 standard deviation increase in the GRS increases the likelihood of being obese by 5.2 percentage points. The GxE interactions are statistically insignificant, suggesting that the marginal association of the GRS with obesity does not differ by educational attainment. When controlling for confounding through twins fixed-effects, we find a smaller role of genetics. The association of the GRS with obesity decreases to 0.040. Associations of educational attainment are substantially reduced and insignificant. GxE interactions also remain insignificant. Overall, we find little evidence of any GxE interactions.

Next Generation Sequencing Methods for Diagnosis of Epilepsy Syndromes.

Epilepsy is a neurological disorder characterized by an increased predisposition for seizures. Although this definition suggests that it is a single disorder, epilepsy encompasses a group of disorders with diverse aetiologies and outcomes. A genetic basis for epilepsy syndromes has been postulated for several decades, with several mutations in specific genes identified that have increased our understanding of the genetic influence on epilepsies. With 70-80% of epilepsy cases identified to have a genetic cause, there are now hundreds of genes identified to be associated with epilepsy syndromes which can be analyzed using next generation sequencing (NGS) techniques such as targeted gene panels, whole exome sequencing (WES) and whole genome sequencing (WGS). For effective use of these methodologies, diagnostic laboratories and clinicians require information on the relevant workflows including analysis and sequencing depth to understand the specific clinical application and diagnostic capabilities of these gene sequencing techniques. As epilepsy is a complex disorder, the differences associated with each technique influence the ability to form a diagnosis along with an accurate detection of the genetic etiology of the disorder. In addition, for diagnostic testing, an important parameter is the cost-effectiveness and the specific diagnostic outcome of each technique. Here, we review these commonly used NGS techniques to determine their suitability for application to epilepsy genetic diagnostic testing.

Anti-N-methyl-d-aspartate receptor encephalitis in Maori and Pacific Island children in New Zealand.

AIM: To investigate the incidence and severity of anti-N-methyl-d-aspartate (anti-NMDA) receptor encephalitis in children from New Zealand. METHOD: A retrospective case series was undertaken of all children (≤18y) diagnosed with anti-NMDA receptor encephalitis from January 2008 to October 2015. RESULTS: Sixteen patients were identified with anti-NMDA receptor antibodies in the cerebrospinal fluid, three of whom had an associated teratoma. Fifteen children had Maori and/or Pacific Island ancestry. The incidence of anti-NMDA receptor encephalitis in Maori children was 3.4 per million children per year (95% confidence interval [CI] 1.4-7.0) and the incidence in Pacific children was 10.0 per million children per year (95% CI 4.3-19.8) compared with 0.2 per million children per year (95% CI 0.0-1.0) in children without Maori or Pacific Island ancestry. Sixty-seven per cent of children had a good outcome (modified Rankin Score ≤2) at 2 years' follow-up. This compares unfavourably with other cohorts despite a shorter median time to first-line immunotherapy (13d; range 4-89) and a higher proportion of children being treated with second-line therapy (50%). INTERPRETATION: Maori and Pacific Island children have a higher incidence of anti-NMDA receptor encephalitis and possibly a more severe phenotype. These data suggest a genetic predisposition to anti-NMDA receptor encephalitis in these populations.

Unambiguous high resolution genotyping of human leukocyte antigens.

We have developed a high resolution sequencing based typing method for genotyping Human Leukocyte Antigens (HLA) over a period of twenty years. The methods are based upon the separation of HLA alleles per locus at the initial amplification to simplify the analysis post-sequencing. The increasing discovery of polymorphism in HLA, manifested in new alleles, has necessitated the continuing development of this method. Here we present methods for the high resolution Sequence Based Typing of HLA-A, B, C (class I) and HLA-DQB1 and DRB1 (class II). The purpose of this article is to provide a valuable resource of methods and primers for other laboratories engaged in HLA typing.

Diversity in exon 5 of HLA-C(∗)04:01:01G is significant in anthropological studies.

Polymorphisms in Human Leukocyte Antigen (HLA) class I genes are generally considered to be relevant only if they reside in exons 2 and 3 or if they affect the expression of the allele. HLA-C(∗)04:82 differs from the common HLA-C(∗)04:01:01 by having a 9 nucleotide, or 3 amino acid duplication, in exon 5. Having observed HLA-C(∗)04:82 in a New Zealand Maori stem cell patient, we have attempted to examine the prevalence of this allele in different ethnicities. Although our studies are in a limited number of patients and donors, they have revealed that, in the Pacific region, HLA-C(∗)04:82 appears to be the most common allele of the HLA-C(∗)04:01:01G group of alleles, notably in Filippinos and in Maori/Polynesians. In these populations this allele has characteristic HLA-ABCDRB1 haplotypes. Thus, our studies have shown that polymorphisms outside of the clinically important exons can be considered to be relevant in anthropological studies.

Recent Developments in Transplantation and Transfusion Medicine.

Transplantation and transfusion are related and clinically important areas of multidisciplinary expertise, including pre-operative treatment, donor recruitment, tissue matching, and post-operative care. We have seen significant developments in these areas, especially in the late 20th and early 21st century. This paper reviews the latest advances in modern transplantation and transfusion medicine, including several new genetic markers (e.g., major histocompatibility complex class I chain-related gene A, killer cell immunoglobulin-like receptor, and human platelet antigens) for donor and recipient matching, genotyping platforms (e.g., next-generation sequencer and Luminex technology), donor recruitment strategies, and several clinical applications in which genotyping has advantages over agglutination tests (e.g., genotyping of weakly expressed antigens and determination of blood groups and human leukocyte antigen types in multi-transfused patients). We also highlight the roles of population studies and international collaborations in moving towards more efficient donor recruitment strategies.

Novel Approaches and Technologies in Molecular HLA Typing.

The invention of the Polymerase Chain Reaction (PCR) has revolutionized molecular biology enabling gene isolation and characterization in hours rather than days. Scientists working in transplant diagnostics have proven to be pioneers in adapting this molecular technique to the clinical needs of histocompatibility testing. This chapter describes a number of novel genotyping technologies which have been used to address the challenges posed by genetic diversity seen in the extensive polymorphism in HLA genes. These novel approaches include single-stranded and duplex conformational analyses, real-time PCR, microarray hybridization, RNA-based sequencing, and the present day Next Generation Sequencing. The chapter concludes with a brief look at a possible next, Next Generation Sequencing system.

Coelomic Transport and Clearance of Durable Foreign Bodies by Starfish (Asterias rubens).

Echinoderms have excellent healing and regeneration abilities, but little is known about how they deal with the related challenge of durable foreign bodies that become lodged within their bodies. Here we report a novel mechanism for foreign body elimination in starfish. When injected into the arm of a starfish, passive integrated transponder tags and magnets of similar dimensions are eliminated at a rate approximating 10% per day. These objects are forcefully ejected through the body wall at the distal tip of an arm. Ultrasound images reveal that foreign bodies are moved within the body cavity, and tracking of magnets injected into starfish suggests that the movements are haphazard rather than directed. Constrictions of the body wall near the foreign object are the likely mechanism for this transport process. Open questions include the ecological relevance of this behavior, why clearance occurs through the distal tips of the arms, the neurological and muscular control of this behavior, what other animals use this mechanism, and the range of objects starfish can eliminate in this way.

Salinity and Temperature Tolerance of the Nemertean Worm Carcinonemertes errans, an Egg Predator of the Dungeness Crab.

Estuaries can be harsh habitats for the marine animals that enter them, but they may also provide these species with sub-saline refuges from their parasites. The nemertean egg predator Carcinonemertes errans is known to occur less frequently and in smaller numbers on its host, the Dungeness crab Metacarcinus magister, when the hosts are found within estuaries. We examined the temperature and salinity tolerances of C. errans to determine if this observed distribution represents a true salinity refuge. We monitored the survival of juvenile and larval worms exposed to ecologically relevant salinities (5-30) and temperatures (8-20 °C) over the course of several days under laboratory conditions. Juvenile worms were unaffected by the experimental temperature levels and exhibited robustness to salinity treatments 25 and 30. However, significant mortality was seen at salinity treatments 20 and below. Larvae were less tolerant than juveniles to lowered salinity and were also somewhat more susceptible to the higher temperatures tested. Given that the Dungeness crab can tolerate forays into mesohaline (salinity 5-18) waters for several days at a time, our findings suggest that salinity gradients play an important role in creating a parasite refuge for this species within the estuaries of the Pacific Northwest.

KIR diversity in Maori and Polynesians: populations in which HLA-B is not a significant KIR ligand.

HLA class I molecules and killer cell immunoglobulin-like receptors (KIR) form a diverse system of ligands and receptors that individualize human immune systems in ways that improve the survival of individuals and populations. Human settlement of Oceania by island-hopping East and Southeast Asian migrants started ~3,500 years ago. Subsequently, New Zealand was reached ~750 years ago by ancestral Maori. To examine how this history impacted KIR and HLA diversity, and their functional interaction, we defined at high resolution the allelic and haplotype diversity of the 13 expressed KIR genes in 49 Maori and 34 Polynesians. Eighty KIR variants, including four 'new' alleles, were defined, as were 35 centromeric and 22 telomeric KIR region haplotypes, which combine to give >50 full-length KIR haplotypes. Two new and divergent variant KIR form part of a telomeric KIR haplotype, which appears derived from Papua New Guinea and was probably obtained by the Asian migrants en route to Polynesia. Maori and Polynesian KIR are very similar, but differ significantly from African, European, Japanese, and Amerindian KIR. Maori and Polynesians have high KIR haplotype diversity with corresponding allotype diversity being maintained throughout the KIR locus. Within the population, each individual has a unique combination of HLA class I and KIR. Characterizing Maori and Polynesians is a paucity of HLA-B allotypes recognized by KIR. Compensating for this deficiency are high frequencies (>50 %) of HLA-A allotypes recognized by KIR. These HLA-A allotypes are ones that modern humans likely acquired from archaic humans at a much earlier time.