Low incidence of BRAF and NRAS mutations in a population with a high incidence of melanoma.

Reported rates of BRAF mutation in Irish cutaneous melanoma cohorts are lower than the reported international data. We aimed to assess the mutational status of a cohort of primary cutaneous melanomas and to correlate it with clinical follow-up data.A total of 92 cases of primary cutaneous melanoma diagnosed at a single institution in 2012 were analyzed. Regions containing common mutations in the BRAF, NRAS, KIT, and KRAS genes were investigated by PCR amplification followed by Sanger sequencing. Demographic details, tumor characteristics, and 10-year outcome data were also obtained.Ten cases with BRAF V600E mutations (11.6%) and five (5.49%) NRAS mutations (4 at Q61R, 1 at Q61K) were detected. No statistically significant differences were noted between groups for age, gender, depth of invasion, nodal status, or recurrence status (p ≥ 0.05).These findings suggest that the Irish population has a markedly lower incidence of BRAF and NRAS mutations in melanoma than those reported in other cohorts.

Implementation of an ISO 15189 accredited next generation sequencing service for cell-free total nucleic acid (cfTNA) analysis to facilitate driver mutation reporting in blood: the experience of a clinical diagnostic laboratory.

AIMS: Next generation sequencing (NGS) on tumour tissue is integral to the delivery of personalised medicine and targeted therapy. NGS on liquid biopsy, a much less invasive technology, is an emerging clinical tool that has rapidly expanded clinical utility. Gene mutations in cell-free total nucleic acids (cfTNA) circulating in the blood are representative of whole tumour biology and can reveal different mutations from different tumour sites, thus addressing tumour heterogeneity challenges. METHODS: The novel Ion Torrent Genexus NGS system with automated sample preparation, onboard library preparation, templating, sequencing, data analysis and Oncomine Reporter software was used. cfTNA extracted from plasma was verified with the targeted pan-cancer (~50 genes) Oncomine Precision Assay (OPA). Assessment criteria included analytical sensitivity, specificity, limits of detection (LOD), accuracy, repeatability, reproducibility and the establishment of performance metrics. RESULTS: An ISO 15189 accredited, minimally invasive cfTNA NGS diagnostic service has been implemented. High sensitivity (>83%) and specificity between plasma and tissue were observed. A sequencing LOD of 1.2% was achieved when the depth of coverage was >22 000×. A reduction (>68%) in turnaround time (TAT) of liquid biopsy results was achieved: 5 days TAT for in-house analysis from sample receipt to a final report issued to oncologists as compared with >15 days from reference laboratories. CONCLUSION: Tumour-derived somatic variants can now be reliably assessed from plasma to provide minimally invasive tumour profiling. Successful implementation of this accredited service resulted in:Appropriate molecular profiling of patients where tumour tissue is unavailable or inaccessible.Rapid TAT of plasma NGS results.

A novel locus for restless legs syndrome maps to chromosome 19p in an Irish pedigree.

Restless legs syndrome (RLS) is a common, sleep-related movement disorder. The symptoms follow a circadian pattern, worsening in the evening or night, leading to sleep disruption and daytime somnolence. Familial forms of RLS have been described and usually display an autosomal dominant pattern of inheritance. To date, linkage analysis has identified nine RLS loci, but no specific causative gene has been reported. Association mapping has highlighted a further four genomic areas of interest. We have conducted a genome-wide linkage analysis in an Irish autosomal dominant RLS pedigree with 11 affected members. Significant linkage was found on chromosome 19p for a series of microsatellite markers, with a maximum two-point LOD score of 3.59 at θ = 0.0 for marker D19S878. Recombination events, identified by haplotype analysis, define a genetic region of 6.57 cM on chromosome 19p13.3, corresponding to an interval of 2.5 Mb. This study provides evidence of a novel RLS locus and provides further evidence that RLS is a genetically heterogenous disorder.

Implementation of an ISO15189 accredited next-generation sequencing service with the fully automated Ion Torrent Genexus: the experience of a clinical diagnostic laboratory.

AIMS: Next-generation sequencing (NGS) is integral to the delivery of personalised medicine for targeted cancer therapy. Average turnaround times (TAT) from reference laboratories with advanced expertise in sequencing are typically 2-3 weeks. Prolonged TAT for biomarker analysis can adversely affect patient outcomes. The project aim was to establish an accredited NGS service integrated within a routine clinical diagnostic laboratory, in a designated tertiary cancer centre with no previous experience in NGS or bioinformatics. METHODS: Platform selected was the novel Ion Torrent Genexus Sequencer with automated onboard library preparation, templating, sequencing and data analysis, with subsequent reporting using Oncomine Reporter software.Entire workflow validation was performed with a targeted panel, the Oncomine Precision Assay, on formalin-fixed paraffin embedded clinical tumour samples. Oncomine Reporter software was used to report on variants including mutations, copy number variations and fusions across 50 key genes.Samples included surgical resections, biopsies, cytology and commercial reference material. Assessment of criteria included analytical sensitivity, specificity, limit of detection, accuracy, repeatability and reproducibility, with the establishment of performance metrics and quality parameters. RESULTS: High sensitivity, specificity and reproducibility were achieved. DNA/RNA input requirements optimised to >10 ng, and sequencing performance established with a limit of detection of 5% when depth of coverage of 2500X was reached. This NGS service attained ISO15189 accreditation with no non-conformances and >56% reduction in TAT. CONCLUSION: Successful implementation, clinical validation and accreditation of a novel NGS technology was achieved in this institution, with a significantly improved TAT of results to oncologists.

Mutagenesis by Microbe: the Role of the Microbiota in Shaping the Cancer Genome.

Cancers arise through the process of somatic evolution fueled by the inception of somatic mutations. We lack a complete understanding of the sources of these somatic mutations. Humans host a vast repertoire of microbes collectively known as the microbiota. The microbiota plays a role in altering the tumor microenvironment and proliferation. In addition, microbes have been shown to elicit DNA damage which provides the driver for somatic mutations. An understanding of microbiota-driven mutational mechanisms would contribute to a more complete understanding of the origins of the cancer genome. Here, we review the modes by which microbes stimulate DNA damage and the effect of these phenomena upon the cancer genomic architecture, specifically in the form of mutational spectra and mutational signatures.

Homozygous SLC4A11 mutation in a large Irish CHED2 pedigree.

BACKGROUND: Congenital hereditary endothelial dystrophy (CHED) is a genetic disorder of corneal endothelial cells resulting in corneal clouding and visual impairment. Autosomal dominant (CHED1) and autosomal recessive (CHED2) forms have been reported and map to distinct loci on chromosome 20. CHED2 is caused by mutations in the SLC4A11 gene which encodes a membrane transporter protein. MATERIALS AND METHODS: Members of a large CHED2 family were recruited for clinical and genetic studies. Genomic DNA was sequenced for the exons and intron-exon boundaries of the SLC4A11 gene. RESULTS: Twelve family members were recruited, of which eight were diagnosed with CHED. A homozygous SLC4A11 mutation (Leu843Pro) was detected in the eight patients; a single copy of the mutation was present in three unaffected carriers. CONCLUSIONS: A missense SLC4A11 mutation (Leu843Pro) is responsible for CHED2 in this family; this is the first report of this mutation in a homozygous state.

Spectrum of SPG4 mutations in a large collection of North American families with hereditary spastic paraplegia.

BACKGROUND: Hereditary spastic paraplegia (HSP) is a neurodegenerative disease characterized by progressive spasticity and weakness of the lower limbs. The most common form of HSP is caused by mutations in the SPG4 gene, which codes for spastin, an adenosine triphosphatase with various cellular activities (AAA) protein family member. OBJECTIVE: To investigate a large collection of predominantly North American patients with HSP for mutations in the spastin encoding gene, SPG4. METHODS: DNA from 76 unrelated affected individuals was studied for mutations by single-stranded conformational polymorphism analysis and direct sequencing. Each new variant identified was then analyzed in 80 control subjects to determine whether the variant is a common polymorphism or a rare mutation. All DNA samples were amplified by polymerase chain reaction, followed by electrophoresis and autoradiography. RESULTS: We identified 8 novel mutations and 5 previously reported mutations in 15 affected individuals. The novel mutations are 4 missense, 1 nonsense, 1 frameshift, and 2 splice mutations. Two polymorphisms (one in an affected individual) were also identified. CONCLUSIONS: Our collection of families with HSP is different on a genetic level from those previously described. The percentage of our families with a SPG4 mutation is 10% lower than the 40% estimate of families with autosomal dominant HSP noted to be linked to this locus, and splice mutations are not predominant in our collection. Interestingly, we also identified 2 recurring mutations in specific populations (R562Q and G559D), which may facilitate the development of future spastin diagnostic testing in these populations.

Mutation screening of the ALS2 gene in sporadic and familial amyotrophic lateral sclerosis.

BACKGROUND: Mutations in the ALS2 gene cause juvenile-onset autosomal recessive amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia. OBJECTIVE: To assess the role of ALS2 among more common forms of ALS. METHODS: DNA from 95 unrelated familial, 95 unrelated sporadic, and 11 early-onset ALS patients was screened for mutations in ALS2 by denaturing high-performance liquid chromatography and direct sequencing of polymerase chain reaction-amplified fragments. Each variant identified was also analyzed among control subjects. All 34 exons of ALS2 plus the 5' and 3' untranslated region were screened. RESULTS: We detected 23 novel sequence variants; however, none is disease-associated. CONCLUSION: Mutations of ALS2 are not a common cause of ALS.

Novel RPGR mutations with distinct retinitis pigmentosa phenotypes in French-Canadian families.

PURPOSE: To characterize the molecular defects in two x-linked retinitis pigmentosa (RP) families. We hypothesized that different RPGR mutations result in distinct RP phenotypes. DESIGN: Observational case series. METHODS: Fifteen members in family I and three members in family II were evaluated. Full ophthalmic evaluations were done. Linkage analyses were performed and likelihood of odds scores (LOD score) were calculated. For mutation analyses, we used dHPLC and automated sequencing. RESULTS: Two novel RPGR mutations were identified in the two families; a Glu 414 (2-bp del) frameshift mutation in family I and an IVS 2-1 (g to a) splice site mutation in family II. All male family members in family I were severely affected by RP but maintained central visual acuities until their 50s and did not develop a bull's eye maculopathy. The female phenotype was highly variable. Some of the carriers exhibited a severe phenotype, one female displayed an asymmetric phenotype, and other carriers were asymptomatic. All members with the RPGR frameshift mutation exhibited rod-cone electroretinograms abnormalities, whereas five members had hearing loss. Male members of family II were severely affected, with early visual acuity loss, central scotomas, and bull's eye maculopathy. The female family members were asymptomatic but displayed cone-rod electroretinograms changes. There was no hearing loss. CONCLUSIONS: Different RPGR mutations lead to distinct RP phenotypes, with a highly variable inter- and intrafamilial phenotypic spectrum of disease that is associated with the type of mutation in RPGR and nonrandom X chromosome inactivation, respectively.

Linkage to the CCM2 locus and genetic heterogeneity in familial cerebral cavernous malformation.

BACKGROUND: Cerebral cavernous malformation (CCM) is a form of intracranial vascular disease that may arise sporadically or be dominantly inherited. Linkage studies have revealed genetic heterogeneity among the dominantly inherited forms suggesting the existence of at least three loci called CCM1, CCM2 and CCM3. METHODS: In the present study, we screened five families with dominantly inherited CCM for CCM1 gene mutations with denaturing high performance liquid chromatography (DHPLC). Then, we performed linkage analysis and haplotyping on these five families using highly polymorphic markers at the candidate CCM loci. RESULTS: None of the five families tested with DHPLC were found to have mutations in the CCM1 gene. Based on haplotyping, we identified three families segregating alleles for CCM2, while two families segregated alleles for CCM3. Using linkage analysis, we could confirm that one family (IFCAS-1) had a positive Lod score of 2.03 (p<0.0001) at the CCM2 locus using marker D7S678. CONCLUSIONS: The present study is the first one to replicate linkage at the CCM2 locus and provides a fifth family identified as such. It also supports the concept of genetic heterogeneity in CCM, identifying four other families that showed no mutations in the CCM1 gene.

Andersen-Tawil syndrome with early fixed myopathy.

Andersen-Tawil syndrome (ATS) is a rare autosomal dominant potassium channelopathy characterized by a triad of periodic paralysis, ventricular arrhythmias, and distinctive dysmorphic abnormalities. We present a 19-year-old man with characteristic skeletal dysmorphic features of ATS, early nonfluctuating proximal lower limb weakness from childhood, and neonatal focal seizures. He later developed fluctuating weakness in addition to a fixed proximal myopathy. A 12-lead electrocardiogram showed prominent "U" waves, and McManis protocol prolonged exercise test showed an unusually early decline in the compound motor action potential amplitude by 51%. Genetic testing revealed a de novo heterozygous mutation (R218W) in KCNJ2 associated with ATS. This is the first reported case of ATS in an Irish population with an unusual fixed myopathy from early childhood.

A novel ATP1A2 gene mutation in an Irish familial hemiplegic migraine kindred.

OBJECTIVE: We studied a large Irish Caucasian pedigree with familial hemiplegic migraine (FHM) with the aim of finding the causative gene mutation. BACKGROUND: FHM is a rare autosomal-dominant subtype of migraine with aura, which is linked to 4 loci on chromosomes 19p13, 1q23, 2q24, and 1q31. The mutations responsible for hemiplegic migraine have been described in the CACNA1A gene (chromosome 19p13), ATP1A2 gene (chromosome 1q23), and SCN1A gene (chromosome 2q24). METHODS: We performed linkage analyses in this family for chromosome 1q23 and performed mutation analysis of the ATP1A2 gene. RESULTS: Linkage to the FHM2 locus on chromosome 1 was demonstrated. Mutation screening of the ATP1A2 gene revealed a G to C substitution in exon 22 resulting in a novel protein variant, D999H, which co-segregates with FHM within this pedigree and is absent in 50 unaffected individuals. This residue is also highly conserved across species. CONCLUSIONS: We propose that D999H is a novel FHM ATP1A2 mutation.

Familial amyotrophic lateral sclerosis.

The increasing complexity of the pathways implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS) has stimulated intensive research in many directions. Genetic analysis of familial ALS has yielded six loci and one disease gene (SOD1), initially suggesting a role for free radicals in the disease process, although the mechanisms through which the mutant exerts toxicity and results in selective motor neuron death remain uncertain. Numerous studies have focused on structural elements of the affected cell, emphasizing the role of neurofilaments and peripherin and their functional disruption in disease. Other topics examined include cellular homeostasis of copper and calcium, particularly in the context of oxidative stress and the processes of protein aggregation, glutamate excitotoxicity, and apoptosis. It has become evident that there is considerable interplay between these mechanisms and, as the role of each is established, a common picture may emerge, enabling the development of more targeted therapies. This study discusses the main areas of investigation and reviews the findings.

A novel locus for familial amyotrophic lateral sclerosis, on chromosome 18q.

Amyotrophic lateral sclerosis (ALS) is an adult-onset degenerative disorder characterized by the death of motor neurons in the cortex, brain stem, and spinal cord. Despite intensive research the basic pathophysiology of ALS remains unclear. Although most cases are sporadic, approximately 10% of ALS cases are familial (FALS). Mutations in the Cu/Zn superoxide dismutase (SOD1) gene cause approximately 20% of FALS. The gene(s) responsible for the remaining 80% of FALS remain to be found. Using a large European kindred without SOD1 mutation and with classic autosomal dominant adult-onset ALS, we have identified a novel locus by performing a genome scan and linkage analysis. The maximum LOD score is 4.5 at recombination fraction 0.0, for polymorphism D18S39. Haplotype analysis has identified a 7.5-cM, 8-Mb region of chromosome 18q21, flanked by markers D18S846 and D18S1109, as a novel FALS locus.