Specifications of the ACMG/AMP Variant Classification Guidelines for Germline DICER1 Variant Curation.

Germline pathogenic variants in DICER1 predispose individuals to develop a variety of benign and malignant tumors. Accurate variant curation and classification is essential for reliable diagnosis of DICER1-related tumor predisposition and identification of individuals who may benefit from surveillance. Since 2015, most labs have followed the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) sequence variant classification guidelines for DICER1 germline variant curation. However, these general guidelines lack gene-specific nuances and leave room for subjectivity. Consequently, a group of DICER1 experts joined ClinGen to form the DICER1 and miRNA-Processing Genes Variant Curation Expert Panel (VCEP), to create DICER1- specific ACMG/AMP guidelines for germline variant curation. The VCEP followed the FDA-approved ClinGen protocol for adapting and piloting these guidelines. A diverse set of 40 DICER1 variants were selected for piloting, including 14 known Pathogenic/Likely Pathogenic (P/LP) variants, 12 known Benign/Likely Benign (B/LB) variants, and 14 variants classified as variants of uncertain significance (VUS) or with conflicting interpretations in ClinVar. Clinically meaningful classifications (i.e., P, LP, LB, or B) were achieved for 82.5% (33/40) of the pilot variants, with 100% concordance among the known P/LP and known B/LB variants. Half of the VUS or conflicting variants were resolved with four variants classified as LB and three as LP. These results demonstrate that the DICER1-specific guidelines for germline variant curation effectively classify known pathogenic and benign variants while reducing the frequency of uncertain classifications. Individuals and labs curating DICER1 variants should consider adopting this classification framework to encourage consistency and improve objectivity.

Demographic and socioeconomic trends in DNA banking utilization in the USA.

Demographic and clinical information from de-identified individuals utilizing a single DNA banking service over a 22-year period was assessed using descriptive statistics. The socioeconomic characteristics of the study population were estimated using a zip code-level analysis of US Census data and compared to national US Metrics for 2016. Samples from 4,874 individuals were deposited to a single commercial DNA bank from 1997 to 2019. Samples originated from 31 countries across 6 continents, with the majority of samples originating from the United States (US; 97.37%; n = 4,746). A higher proportion of individuals identifying as females (55.58%; n = 2,709) utilized the service compared to males (41.18%; n = 2,007). The age distribution was bimodal, peaking around 5 years of age and again around 65 years of age. Whole blood was the preferred specimen for submission. Sample deposits peaked in 2015 with 559 annual deposits. Clinical genetic counselors were the most common referral source (41.73%; n = 2,034). Individuals utilizing DNA banking services are estimated to reside in wealthier, more educated and less racially diverse zip codes compared to national metrics. Although direct to consumer DNA banking is being utilized by the general public and clinical genetic counselors in the US, it is not widespread.

Family history of breast cancer in men with non-BRCA male breast cancer: implications for cancer risk counseling.

PURPOSE: The role of genetic predisposition in male breast cancer (MBC) patients who test negative for a BRCA mutation is unclear. The aim of this study is to define the association between MBC and family history of breast cancer in patients without mutations in BRCA1 or BRCA2. METHODS: We conducted an unmatched case-control study with men who received commercial testing for germline mutations in cancer susceptibility genes, including 3,647 MBC cases who tested negative for deleterious mutations in BRCA1/BRCA2, and 4,269 men with a personal history of colorectal cancer who tested negative for mutations in DNA mismatch repair genes to serve as controls. Associations between family history of breast cancer and MBC were estimated using unconditional multivariable logistic regression with adjustment for age, race/ethnicity and year of testing. RESULTS: Breast cancer in a first- or second-degree relative was associated with a four-fold increased odds of MBC (OR 4.7; 95% CI 4.1, 5.3). Associations with MBC were strongest for family history of breast cancer in 2 or more first-degree relatives (FDR) (OR 7.8; 95% CI 5.2, 11.6), for probands and FDR diagnosed at age < 45 years (OR 6.9; 95% CI 3.9, 12.4), and for family history of MBC (OR 17.9; 95% CI 7.6, 42.1). Findings were confirmed in a sensitivity analysis of MBC cases who tested negative on a 25-gene pan-cancer panel. CONCLUSIONS: MBC patients without mutations in BRCA1/2 have significantly higher odds of a family history of breast cancer, suggesting the existence of unidentified MBC susceptibility alleles.

A substantial proportion of apparently heterozygous TP53 pathogenic variants detected with a next-generation sequencing hereditary pan-cancer panel are acquired somatically.

Previous analysis of next-generation sequencing (NGS) hereditary pan-cancer panel testing demonstrated that approximately 40% of TP53 pathogenic and likely pathogenic variants (PVs) detected have NGS allele frequencies between 10% and 30%, indicating that they likely are acquired somatically. These are seen more frequently in older adults, suggesting that most result from normal aging-related clonal hematopoiesis. For this analysis, apparent heterozygous germline TP53 PV carriers (NGS allele frequency 30-70%) were offered follow-up testing to confirm variant origin. Ninety-eight probands had samples submitted for follow-up family member testing, fibroblast testing, or both. The apparent heterozygous germline TP53 PV was not detected in 32.6% (15/46) of submitted fibroblast samples, indicating that it was acquired somatically, either through clonal hematopoiesis or via constitutional mosaicism. Notably, no individuals with confirmed germline or likely germline TP53 PVs met classic Li-Fraumeni syndrome (LFS) criteria, only 41% met Chompret LFS criteria, and 59% met neither criteria, based upon provider-reported personal and family cancer history. Comprehensive reporting of TP53 PVs detected using NGS, combined with follow-up analysis to confirm variant origin, is advised for clinical testing laboratories. These findings underscore the investment required to provide individuals and family members with clinically accurate genetic test results pertaining to their LFS risk.

Prevalence and characteristics of likely-somatic variants in cancer susceptibility genes among individuals who had hereditary pan-cancer panel testing.

Next-generation sequencing (NGS) hereditary pan-cancer panel testing can identify somatic variants, which exhibit lower allele frequencies than do germline variants and may confound hereditary cancer predisposition testing. This analysis examined the prevalence and characteristics of likely-somatic variants among 348,543 individuals tested using a clinical NGS hereditary pan-cancer panel. Variants showing allele frequencies between 10% and 30% were interpreted as likely somatic and identified in 753 (0.22%) individuals. They were most frequent in TP53, CHEK2 and ATM, commonly as C-to-T transitions. Among individuals who carried a likely-somatic variant and reported no personal cancer history, 54.2% (78/144) carried a variant in TP53, CHEK2 or ATM. With a reported cancer history, this percentage increased to 81.1% (494/609), predominantly in CHEK2 and TP53. Their presence was associated with age (OR=3.1, 95% CI 2.5, 3.7; p<0.001) and personal history of cancer (OR=3.3, 95% CI 2.7, 4.0; p<0.001), particularly ovarian cancer. Germline ATM pathogenic variant carriers showed significant enrichment of likely-somatic variants (OR=2.8, 95% CI 1.6, 4.9; p = 0.005), regardless of cancer status. The appearance of likely-somatic variants is consistent with clonal hematopoiesis, possibly influenced by cancer treatment. These findings highlight the precision required of diagnostic laboratories to deliver accurate germline testing results.

Hereditary cancer testing challenges: assembling the analytical pieces to solve the patient clinical puzzle.

Expanded genetic test utilization to guide cancer management has driven the development of larger gene panels and greater diversity in the patient population pursuing testing, resulting in increased identification of atypical or technically challenging genetic findings. To ensure appropriate patient care, it is critical that genetic tests adequately identify and characterize these findings. We describe genetic testing challenges frequently encountered by our laboratory and the methodologies we employ to improve test accuracy for the identification and characterization of atypical genetic findings. While these findings may be individually rare, 15,745 (9%) individuals tested by our laboratory for hereditary cancer risk had an atypical genetic finding, highlighting the importance of employing highly accurate and comprehensive methods in clinical genetic testing.

Prevalence of Variant Reclassification Following Hereditary Cancer Genetic Testing.

Importance: Variant reclassification is an important component of hereditary cancer genetic testing; however, there are few published data quantifying the prevalence of reclassification. Objective: Retrospective cohort study of individuals who had genetic testing from 2006 through 2016 at a single commercial laboratory. Design, Setting, and Participants: A retrospective cohort of individuals who had genetic testing between 2006 and 2016 at a single commercial laboratory was assessed. Variants were classified as benign, likely benign, variant of uncertain significance, likely pathogenic, or pathogenic. Retrospective chart reviews were conducted for patients from the University of Texas Southwestern (UTSW) Medical Center. Exposures: Hereditary cancer genetic testing. Main Outcomes and Measures: Frequency of and time to amended reports; frequency and types of variant reclassification. Results: From 2006 through 2018, 1.45 million individuals (median [interquartile range] age at testing, 49 years [40.69-58.31 years], 95.6% women) had genetic testing, and 56.6% (n = 821 724) had a personal history of cancer. A total of 1.67 million initial tests were reported and 59 955 amended reports were issued due to variant reclassification. Overall, 6.4% (2868 of 44 777) of unique variants were reclassified. Reclassification to a different clinical category was rare among unique variants initially classified as pathogenic or likely pathogenic (0.7%, 61 of 9112) or benign or likely benign (0.2%, 15 of 8995). However, 7.7% (2048 of 26 670) of unique variants of uncertain significance were reclassified: 91.2% (1867 of 2048) were downgraded to benign or likely benign (median time to amended report, 1.17 years), 8.7% (178 of 2048) were upgraded to pathogenic or likely pathogenic variants (median time to amended report, 1.86 years). Because most variants were observed in more than 1 individual, 24.9% (46 890 of 184 327) of all reported variants of uncertain significance were reclassified. Conclusions and Relevance: Following hereditary cancer genetic testing at a single commercial laboratory, 24.9% of variants of uncertain significance were reclassified, which included both downgrades and upgrades. Further research is needed to assess generalizability of the findings for other laboratories, as well as the clinical consequences of the reclassification as a component of a genetic testing program.

Identification of pathogenic retrotransposon insertions in cancer predisposition genes.

Cancer risks have been previously reported for some retrotransposon element (RE) insertions; however, detection of these insertions is technically challenging and very few oncogenic RE insertions have been reported. Here we evaluate RE insertions identified during hereditary cancer genetic testing using a comprehensive testing strategy. Individuals who had single-syndrome or pan-cancer hereditary cancer genetic testing from February 2004 to March 2017 were included. RE insertions were identified using Sanger sequencing, Next Generation Sequencing, or multiplex quantitative PCR, and further characterized using targeted PCR and sequencing analysis. Personal cancer history, ancestry, and haplotype were evaluated. A total of 37 unique RE insertions were identified in 10 genes, affecting 211 individuals. BRCA2 accounted for 45.9% (17/37) of all unique RE insertions. Several RE insertions were detected with high frequency in populations of conserved ancestry wherein up to 100% of carriers shared a high degree of haplotype conservation, suggesting founder effects. Our comprehensive testing strategy resulted in a substantial increase in the number of reported oncogenic RE insertions, several of which may have possible founder effects. Collectively, these data show that the detection of RE insertions is an important component of hereditary cancer genetic testing and may be more prevalent than previously reported.

Detection of somatic variants in peripheral blood lymphocytes using a next generation sequencing multigene pan cancer panel.

Next Generation Sequencing (NGS) multigene panels, which are routinely used to assess hereditary cancer risk, can detect both inherited germline variants and somatic variants in cancer-risk genes. We evaluated the frequency and distribution of likely somatic Pathogenic and Likely Pathogenic variants (PVs) detected in >220,000 individuals who underwent clinical testing with a 25-gene panel between September 2013 and March 2016. Likely somatic PVs are defined as variants with NGS read frequencies from 10% to 30%. Overall, 137 (0.06%) individuals were identified as carrying likely somatic PVs, most commonly in TP53 (73), CHEK2 (27), and ATM (20). Among this group, a second PV with a NGS read frequency consistent with a germline variant within the same gene or a different gene on the panel was detected in 21 individuals (15.3%), which is similar to the detection rate in our general testing population. Likely somatic PVs accounted for 38.8% of all PVs in TP53. In comparison, likely somatic PVs accounted for <1% of PVs in most other genes. Likely somatic PVs were more frequently identified in older individuals (p < 0.001). Additional studies are ongoing to further investigate the incidence and clinical implications of somatic variants, enabling the appropriate medical management for these patients.

Assessment of in silico protein sequence analysis in the clinical classification of variants in cancer risk genes.

Missense variants represent a significant proportion of variants identified in clinical genetic testing. In the absence of strong clinical or functional evidence, the American College of Medical Genetics recommends that these findings be classified as variants of uncertain significance (VUS). VUSs may be reclassified to better inform patient care when new evidence is available. It is critical that the methods used for reclassification are robust in order to prevent inappropriate medical management strategies and unnecessary, life-altering surgeries. In an effort to provide evidence for classification, several in silico algorithms have been developed that attempt to predict the functional impact of missense variants through amino acid sequence conservation analysis. We report an analysis comparing internally derived, evidence-based classifications with the results obtained from six commonly used algorithms. We compiled a dataset of 1118 variants in BRCA1, BRCA2, MLH1, and MSH2 previously classified by our laboratory's evidence-based variant classification program. We compared internally derived classifications with those obtained from the following in silico tools: Align-GVGD, CONDEL, Grantham Analysis, MAPP-MMR, PolyPhen-2, and SIFT. Despite being based on similar underlying principles, all algorithms displayed marked divergence in accuracy, specificity, and sensitivity. Overall, accuracy ranged from 58.7 to 90.8% while the Matthews Correlation Coefficient ranged from 0.26-0.65. CONDEL, a weighted average of multiple algorithms, did not perform significantly better than its individual components evaluated here. These results suggest that the in silico algorithms evaluated here do not provide reliable evidence regarding the clinical significance of missense variants in genes associated with hereditary cancer.

Impact of Payer Constraints on Access to Genetic Testing.

BACKGROUND: With increased demand for hereditary cancer genetic testing, some large national health-care insurance payers (LNHPs) have implemented policies to minimize inappropriate testing by mandating consultation with a geneticist or genetic counselor (GC). We hypothesized such a restriction would reduce access and appropriate testing. METHODS: Test cancellation rates (ie, tests ordered that did not result in a reported test result), mutation-positive rates, and turnaround times for comprehensive BRCA1/2 testing for a study LNHP that implemented a GC-mandate policy were determined over the 12 months before and after policy implementation (excluding a 4-month transition period). Cancellation rates were evaluated based on the reason for cancellation, National Comprehensive Cancer Network testing criteria, and self-identified ancestry. A control LNHP was evaluated over the same period for comparison. RESULTS: The study LNHP cancellation rate increased from 13.3% to 42.1% ( P < .001) after policy implementation. This increase was also observed when only individuals who met National Comprehensive Cancer Network criteria for hereditary breast and ovarian cancer testing were considered (9.5% to 37.7%; P < .001). Cancellation rates increased after policy introduction for all ancestries; however, this was more pronounced among individuals of African or Latin American ancestry, for whom cancellation rates rose to 48.9% and 49.6%, respectively, compared with 33.9% for individuals of European ancestry. Over this same time period, control LNHP cancellation rates decreased or stayed the same for all subgroups. CONCLUSION: These findings demonstrate that a GC-mandate policy implemented by a LNHP substantially decreased access to appropriate genetic testing, disproportionately impacting minority populations without any evidence that inappropriate testing was decreased.

Whole-genome haplotyping approaches and genomic medicine.

Genomic information reported as haplotypes rather than genotypes will be increasingly important for personalized medicine. Current technologies generate diploid sequence data that is rarely resolved into its constituent haplotypes. Furthermore, paradigms for thinking about genomic information are based on interpreting genotypes rather than haplotypes. Nevertheless, haplotypes have historically been useful in contexts ranging from population genetics to disease-gene mapping efforts. The main approaches for phasing genomic sequence data are molecular haplotyping, genetic haplotyping, and population-based inference. Long-read sequencing technologies are enabling longer molecular haplotypes, and decreases in the cost of whole-genome sequencing are enabling the sequencing of whole-chromosome genetic haplotypes. Hybrid approaches combining high-throughput short-read assembly with strategic approaches that enable physical or virtual binning of reads into haplotypes are enabling multi-gene haplotypes to be generated from single individuals. These techniques can be further combined with genetic and population approaches. Here, we review advances in whole-genome haplotyping approaches and discuss the importance of haplotypes for genomic medicine. Clinical applications include diagnosis by recognition of compound heterozygosity and by phasing regulatory variation to coding variation. Haplotypes, which are more specific than less complex variants such as single nucleotide variants, also have applications in prognostics and diagnostics, in the analysis of tumors, and in typing tissue for transplantation. Future advances will include technological innovations, the application of standard metrics for evaluating haplotype quality, and the development of databases that link haplotypes to disease.

Origin of the PSEN1 E280A mutation causing early-onset Alzheimer's disease.

BACKGROUND: A mutation in presenilin 1 (E280A) causes early-onset Alzheimer's disease. Understanding the origin of this mutation will inform medical genetics. METHODS: We sequenced the genomes of 102 individuals from Antioquia, Colombia. We applied identity-by-descent analysis to identify regions of common ancestry. We estimated the age of the E280A mutation and the local ancestry of the haplotype harboring this mutation. RESULTS: All affected individuals share a minimal haplotype of 1.8 Mb containing E280A. We estimate a time to most recent common ancestor of E280A of 10 (95% credible interval, 7.2-12.6) generations. We date the de novo mutation event to 15 (95% credible interval, 11-25) generations ago. We infer a western European geographic origin of the shared haplotype. CONCLUSIONS: The age and geographic origin of E280A are consistent with a single founder dating from the time of the Spanish Conquistadors who began colonizing Colombia during the early 16th century.

Association of a GRIA3 gene polymorphism with migraine in an Australian case-control cohort.

BACKGROUND: The excitatory neurotransmitter glutamate has been implicated in both the hyperexcitability required for cortical spreading depression as well as activation of the trigeminovascular system required for the allodynia associated with migraine. Polymorphisms in the glutamate receptor ionotropic amino-3-hydroxy-5-methyl-4-isoxazole-propionin acid 1 (GRIA1) and GRIA3 genes that code for 2 of 4 subunits of the glutamate receptor have been previously associated with migraine in an Italian population. In addition, the GRIA3 gene is coded within a previously identified migraine susceptibility locus at Xq24. This study investigated the previously associated polymorphisms in both genes in an Australian case-control population. METHODS: Variants in GRIA1 and GRIA3 were genotyped in 472 unrelated migraine cases and matched controls, and data were analyzed for association. RESULTS: Analysis showed no association between migraine and the GRIA1 gene. However, association was observed with the GRIA3 single nucleotide polymorphism (SNP) rs3761555 (P=.008). CONCLUSION: The results of this study confirmed the previous report of association at the rs3761555 SNP within the migraine with aura subgroup of migraineurs. However, the study identified association with the inverse allele suggesting that rs3761555 may not be the causative SNP but is more likely in linkage disequilibrium with another causal variant in both populations. This study supports the plethora of evidence suggesting that glutamate dysfunction may contribute to migraine susceptibility, warranting further investigation of the glutamatergic system and particularly of the GRIA3 gene.

Unique X-linked familial FSGS with co-segregating heart block disorder is associated with a mutation in the NXF5 gene.

Focal segmental glomerulosclerosis (FSGS) is the consequence of a disease process that attacks the kidney's filtering system, causing serious scarring. More than half of FSGS patients develop chronic kidney failure within 10 years, ultimately requiring dialysis or renal transplantation. There are currently several genes known to cause the hereditary forms of FSGS (ACTN4, TRPC6, CD2AP, INF2, MYO1E and NPHS2). This study involves a large, unique, multigenerational Australian pedigree in which FSGS co-segregates with progressive heart block with apparent X-linked recessive inheritance. Through a classical combined approach of linkage and haplotype analysis, we identified a 21.19 cM interval implicated on the X chromosome. We then used a whole exome sequencing approach to identify two mutated genes, NXF5 and ALG13, which are located within this linkage interval. The two mutations NXF5-R113W and ALG13-T141L segregated perfectly with the disease phenotype in the pedigree and were not found in a large healthy control cohort. Analysis using bioinformatics tools predicted the R113W mutation in the NXF5 gene to be deleterious and cellular studies support a role in the stability and localization of the protein suggesting a causative role of this mutation in these co-morbid disorders. Further studies are now required to determine the functional consequence of these novel mutations to development of FSGS and heart block in this pedigree and to determine whether these mutations have implications for more common forms of these diseases in the general population.

Investigation of lymphotoxin α genetic variants in migraine.

Migraine is a common neurological disease with a genetic basis affecting approximately 12% of the population. Pain during a migraine attack is associated with activation of the trigeminal nerve system, which carries pain signals from the meninges and the blood vessels infusing the meninges to the trigeminal nucleus in the brain stem. The release of inflammatory mediators following cortical spreading depression (CSD) may further promote and sustain the activation and sensitization of meningeal nociceptors, inducing the persistent throbbing headache characterised in migraine. Lymphotoxin α (LTA) is a cytokine secreted by lymphocytes and is a member of the tumour necrosis factor (TNF) family. Genetic variation with the TNF and LTA genes may contribute to threshold brain excitability, propagation of neuronal hyperexcitability and thus initiation and maintenance of a migraine attack. Three LTA variants rs2009658, rs2844482 and rs2229094 were identified in a recent pGWAS study conducted in the Norfolk Island population as being potentially implicated in migraine with nominally significant p values of p=0.0093, p=0.0088 and p=0.033 respectively. To determine whether these SNPs played a role in migraine in a general outbred population these SNPs were gentoyped in a large case control Australian Caucasian population and tested for association with migraine. All three SNPs showed no association in our cohort (p>0.05). Validation of GWAS data in independent case-controls cohorts is essential to establish risk validity within specific population groups. The importance of cytokines in modulating neural inflammation and pain threshold in addition to other studies showing associations between TNF-α and SNPs in the LTA gene with migraine, suggests that LTA could be an important factor contributing to migraine. Although the present study did not support a role for the tested LTA variants in migraine, investigation of other variants within the LTA gene is still warranted.

An X chromosome association scan of the Norfolk Island genetic isolate provides evidence for a novel migraine susceptibility locus at Xq12.

Migraine is a common and debilitating neurovascular disorder with a complex envirogenomic aetiology. Numerous studies have demonstrated a preponderance of women affected with migraine and previous pedigree linkage studies in our laboratory have identified susceptibility loci on chromosome Xq24-Xq28. In this study we have used the genetic isolate of Norfolk Island to further analyse the X chromosome for migraine susceptibility loci.An association approach was employed to analyse 14,124 SNPs spanning the entire X chromosome. Genotype data from 288 individuals comprising a large core-pedigree, of which 76 were affected with migraine, were analysed. Although no SNP reached chromosome-wide significance (empirical α = 1 × 10(-5)) ranking by P-value revealed two primary clusters of SNPs in the top 25. A 10 SNP cluster represents a novel migraine susceptibility locus at Xq12 whilst a 11 SNP cluster represents a previously identified migraine susceptibility locus at Xq27. The strongest association at Xq12 was seen for rs599958 (OR = 1.75, P = 8.92 × 10(-4)), whilst at Xq27 the strongest association was for rs6525667 (OR = 1.53, P = 1.65 × 10(-4)). Further analysis of SNPs at these loci was performed in 5,122 migraineurs from the Women's Genome Health Study and provided additional evidence for association at the novel Xq12 locus (P<0.05).Overall, this study provides evidence for a novel migraine susceptibility locus on Xq12. The strongest effect SNP (rs102834, joint P = 1.63 × 10(-5)) is located within the 5'UTR of the HEPH gene, which is involved in iron homeostasis in the brain and may represent a novel pathway for involvement in migraine pathogenesis.

A genome-wide analysis of 'Bounty' descendants implicates several novel variants in migraine susceptibility.

Migraine is a common neurological disease with a complex genetic aetiology. The disease affects ~12% of the Caucasian population and females are three times more likely than males to be diagnosed. In an effort to identify loci involved in migraine susceptibility, we performed a pedigree-based genome-wide association study of the isolated population of Norfolk Island, which has a high prevalence of migraine. This unique population originates from a small number of British and Polynesian founders who are descendents of the Bounty mutiny and forms a very large multigenerational pedigree (Bellis et al.; Human Genetics, 124(5):543-5542, 2008). These population genetic features may facilitate disease gene mapping strategies (Peltonen et al.; Nat Rev Genet, 1(3):182-90, 2000. In this study, we identified a high heritability of migraine in the Norfolk Island population (h (2) = 0.53, P = 0.016). We performed a pedigree-based GWAS and utilised a statistical and pathological prioritisation approach to implicate a number of variants in migraine. An SNP located in the zinc finger protein 555 (ZNF555) gene (rs4807347) showed evidence of statistical association in our Norfolk Island pedigree (P = 9.6 × 10(-6)) as well as replication in a large independent and unrelated cohort with >500 migraineurs. In addition, we utilised a biological prioritisation to implicate four SNPs, in within the ADARB2 gene, two SNPs within the GRM7 gene and a single SNP in close proximity to a HTR7 gene. Association of SNPs within these neurotransmitter-related genes suggests a disrupted serotoninergic system that is perhaps specific to the Norfolk Island pedigree, but that might provide clues to understanding migraine more generally.