Further insight into the global variability of the OCA2-HERC2 locus for human pigmentation from multiallelic markers

The OCA2-HERC2 locus is responsible for the greatest proportion of eye color variation in humans. Numerous studies extensively described both functional SNPs and associated patterns of variation over this region. The goal of our study is to examine how these haplotype structures and allelic associations vary when highly variable markers such as microsatellites are used. Eleven microsatellites spanning 357 Kb of OCA2-HERC2 genes are analyzed in 3029 individuals from worldwide populations. We found that several markers display large differences in allele frequency (10% to 35% difference) among Europeans, East Asians and Africans. In Europe, the alleles showing increased frequency can also discriminate individuals with (IrisPlex) predicted blue and brown eyes. Distinct haplotypes are identified around the variants C and T of the functional SNP rs12913832 (associated to blue eyes), with linkage disequilibrium r2 values significant up to 237 Kb. The haplotype carrying the allele rs12913832 C has high frequency (76%) in blue eye predicted individuals (30% in brown eye predicted individuals), while the haplotype associated to the allele rs12913832 T is restricted to brown eye predicted individuals. Finally, homozygosity values reach levels of 91% near rs12913832. Odds ratios show values of 4.2, 7.4 and 10.4 for four markers around rs12913832 and 7.1 for their core haplotype. Hence, this study provides an example on the informativeness of multiallelic markers that, despite their current limited potential contribution to forensic eye color prediction, supports the use of microsatellites for identifying causing variants showing similar genetic features and history.

Founder Effects in Hereditary Hemorrhagic Telangiectasia

A founder effect can result from the establishment of a new population by individuals from a larger population or bottleneck events. Certain alleles may be found at much higher frequencies because of genetic drift immediately after the founder event. We provide a systematic literature review of the sporadically reported founder effects in hereditary hemorrhagic telangiectasia (HHT). All publications from the ACVRL1, ENG and SMAD4 Mutation Databases and publications searched for terms “hereditary hemorrhagic telangiectasia” and “founder” in PubMed and Scopus, respectively, were extracted. Following duplicate removal, 141 publications were searched for the terms “founder” and “founding” and the etymon “ancest”. Finally, 67 publications between 1992 and 2020 were reviewed. Founder effects were graded upon shared area of ancestry/residence, shared core haplotypes, genealogy and prevalence. Twenty-six ACVRL1 and 12 ENG variants with a potential founder effect were identified. The bigger the cluster of families with a founder mutation, the more remarkable is its influence to the populational ACVRL1/ENG ratio, affecting HHT phenotype. Being aware of founder effects might simplify the diagnosis of HHT by establishing local genetic algorithms. Families sharing a common core haplotype might serve as a basis to study potential second-hits in the etiology of HHT.

The CHEK2 Variant C.349A>G Is Associated with Prostate Cancer Risk and Carriers Share a Common Ancestor

The identification of recurrent founder variants in cancer predisposing genes may have important implications for implementing cost-effective targeted genetic screening strategies. In this study, we evaluated the prevalence and relative risk of the CHEK2 recurrent variant c.349A>G in a series of 462 Portuguese patients with early-onset and/or familial/hereditary prostate cancer (PrCa), as well as in the large multicentre PRACTICAL case–control study comprising 55,162 prostate cancer cases and 36,147 controls. Additionally, we investigated the potential shared ancestry of the carriers by performing identity-by-descent, haplotype and age estimation analyses using high-density SNP data from 70 variant carriers belonging to 11 different populations included in the PRACTICAL consortium. The CHEK2 missense variant c.349A>G was found significantly associated with an increased risk for PrCa (OR 1.9; 95% CI: 1.1–3.2). A shared haplotype flanking the variant in all carriers was identified, strongly suggesting a common founder of European origin. Additionally, using two independent statistical algorithms, implemented by DMLE+2.3 and ESTIAGE, we were able to estimate the age of the variant between 2300 and 3125 years. By extending the haplotype analysis to 14 additional carrier families, a shared core haplotype was revealed among all carriers matching the conserved region previously identified in the high-density SNP analysis. These findings are consistent with CHEK2 c.349A>G being a founder variant associated with increased PrCa risk, suggesting its potential usefulness for cost-effective targeted genetic screening in PrCa families.

A novel RFC1 repeat motif (ACAGG) in two Asia-Pacific CANVAS families

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is a progressive late-onset, neurological disease. Recently, a pentanucleotide expansion in intron 2 of RFC1 was identified as the genetic cause of CANVAS. We screened an Asian-Pacific cohort for CANVAS and identified a novel RFC1 repeat expansion motif, (ACAGG)exp, in three affected individuals. This motif was associated with additional clinical features including fasciculations and elevated serum creatine kinase. These features have not previously been described in individuals with genetically-confirmed CANVAS. Haplotype analysis showed our patients shared the same core haplotype as previously published, supporting the possibility of a single origin of the RFC1 disease allele. We analysed data from >26 000 genetically diverse individuals in gnomAD to show enrichment of (ACAGG) in non-European populations.

A Māori specific RFC1 pathogenic repeat configuration in CANVAS, likely due to a founder allele

Cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS) is a recently recognized neurodegenerative disease with onset in mid- to late adulthood. The genetic basis for a large proportion of Caucasian patients was recently shown to be the biallelic expansion of a pentanucleotide (AAGGG)n repeat in RFC1. Here, we describe the first instance of CANVAS genetic testing in New Zealand Māori and Cook Island Māori individuals. We show a novel, possibly population-specific CANVAS configuration (AAAGG)10-25(AAGGG)exp, which was the cause of CANVAS in all patients. There were no apparent phenotypic differences compared with European CANVAS patients. Presence of a common disease haplotype among this cohort suggests this novel repeat expansion configuration is a founder effect in this population, which may indicate that CANVAS will be especially prevalent in this group. Haplotype dating estimated the most recent common ancestor at ∼1430 ce. We also show the same core haplotype as previously described, supporting a single origin of the CANVAS mutation.

Recent Selection on a Class I ADH Locus Distinguishes Southwest Asian Populations Including Ashkenazi Jews

The derived human alcohol dehydrogenase (ADH)1B*48His allele of the ADH1B Arg48His polymorphism (rs1229984) has been identified as one component of an East Asian specific core haplotype that underwent recent positive selection. Our study has been extended to Southwest Asia and additional markers in East Asia. Fst values (Sewall Wright’s fixation index) and long-range haplotype analyses identify a strong signature of selection not only in East Asian but also in Southwest Asian populations. However, except for the ADH2B*48His allele, different core haplotypes occur in Southwest Asia compared to East Asia and the extended haplotypes also differ. Thus, the ADH1B*48His allele, as part of a core haplotype of 10 kb, has undergone recent rapid increases in frequency independently in the two regions after divergence of the respective populations. Emergence of agriculture may be the common factor underlying the evident selection.

Common Variation at 6q25.3 (TULP4) Influences Risk for Arterial Thrombosis in Myeloproliferative Neoplasms

Inherent tendency for thrombosis is a major complication in myeloproliferative neoplasms (MPN). The molecular basis of thrombosis in MPN is not well understood, however, genetic factors have been proposed to play a role. To agnostically investigate the role of common germline variation in MPN thrombophilia, we performed genome-wide association studies (GWAS) in MPN patient cohorts characterized for arterial thrombosis (AT) events. In our discovery cohort (n=383) from Vienna, Austria, 18% of patients have suffered from AT after MPN diagnosis, and 33% of patients showed records for AT events at any time. Of this discovery cohort, a subset of patients (n=302) selected independently from thrombotic status was genotyped on the Affymetrix Genome-Wide SNP 6.0 array platform. After assessment of case-control setup and genotyping quality as standardly implemented in GWAS, confidently genotyped single nucleotide polymorphisms (SNPs) were tested for allelic association with occurrence of AT after MPN diagnosis. We observed an association signal beyond genome-wide statistical significance (P <5x10-8) at chromosome 6q25.3, tagged by rs6455579 and six additional closely related tag-SNPs. The 6q25.3 risk haplotype is common with a minor allele frequency (MAF) of ~8% in the Vienna MPN cohort. The minor allele rs6455579_C correlated with increased risk for AT. We next genotyped the full discovery cohort from Vienna (n=383) and a replication cohort from Pavia, Italy (n=505) using qPCR-based SNP genotyping for rs6455579 and computed odds ratios (ORs) to estimate the effect sizes of the association. In the Vienna cohort, rs6455579 genotype was significantly associated with both "AT after diagnosis" (P =1.90x10-8, ORhet=6.93) and "AT at any time" (P =6.96x10-4, ORhet=3.07). In the Pavia cohort, an association with "AT after diagnosis" could not be observed (P =0.14), however, the association of rs6455579 with "AT at any time" could be reproduced at formal statistical significance (P =7.92x10-3, ORhet=2.55). Moreover, Kaplan-Meier statistics on the Pavia cohort revealed a significant difference in AT-free survival after diagnosis upon rs6455579 genotype (log-rank-test P =0.009; rs6455579 homozygous major vs. heterozygous), underpinning the relevance of disease duration for the impact of the haplotype on MPN-related AT. To gain insight into the physiological mechanisms behind the 6q25.3 risk haplotype, we next evaluated the discovery cohort for correlations of the haplotype with clinical parameters other than AT. We could observe a significant trend for increased white blood cell (WBC) count in CALR mutated (n=90, P =2.50x10-3) but not in JAK2 mutated (n=182, P =0.52) ET and PMF patients carrying the risk allele. CALR mutated patients have been previously reported to exhibit significantly prolonged thrombosis-free survival as compared to JAK2 mutated patients. Thus, our observation indicates that the 6q25.3 germline risk haplotype might impact on WBC count most strongly in a subgroup of patients considered low risk for thrombosis based on the somatic mutational status. The 6q25.3 core haplotype (R2 >0.3) spans ~300 kilobases, covering intergenic sequence as well as promoter and 5' exons of the TULP4 gene. To test for the possibility of a rare coding variant in TULP4 or other more distant genes underlying the association through long-range linkage disequilibrium, we used genotype imputation in conjunction with the 1000 genomes reference panel to infer genotypes on all untyped variants (MAF>1%) in a 5 megabase region centered on the core haplotype. We did not detect any coding variants reflecting the association, and the tag-SNPs from the initial GWAS remained the most strongly associated variants. Causative non-coding genetic variation identified in GWAS is thought to exert its function through differential regulation of specific target genes. Therefore, we evaluated a potential influence of the 6q25.3 risk haplotype on TULP4 gene expression in peripheral blood (The Cancer Genome Atlas (TCGA) LAML dataset, RNA-Seq on 173 acute myeloid leukemia patients). Indeed we detected significantly decreased TULP4 expression in risk haplotype carriers (P =0.029), providing indirect evidence for reduced TULP4 transcript levels impacting on elevated risk for AT in MPN. Further studies will be required to functionally assess the potential role of TULP4 in MPN-related AT. Disclosures Gisslinger: Novartis: Honoraria, Research Funding, Speakers Bureau; Geron: Consultancy; Sanofi Aventis: Consultancy; Janssen Cilag: Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; AOP ORPHAN: Consultancy, Honoraria, Research Funding, Speakers Bureau. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

A Comprehensive Study of the VHL-R200W Chuvash Polycythemia Mutation Reveals a Gradual Dysregulation of the Hypoxia Pathway in Oncogenesis

Context : Germline mutations in genes involved in the hypoxia sensing pathway (VHL, PHD2/EGLN1, HIF2A/EPAS1) predispose patients to erythrocytosis associated with normal or high serum erythropoietin level. The more frequent mutation, VHL-R200W (R200W), has been identified in homozygous carriers with a congenital erythrocytosis named Chuvash polycythemia. Survival in the Chuvash patients was found to be reduced compared to control groups due to higher rates of arterial and venous thromboses, and to haemorrhagic events. Noteworthily, a characteristic of these patients and their parents, heterozygous for the mutation, is the total absence of tumor development as the opposite of heterozygous carriers of other VHL mutations. Indeed, VHL is a tumor suppressor gene and heterozygous carriers of VHLmutations have von Hippel-Lindau disease and are at high risk of multiple tumors (e.g. CNS hemangioblastomas, pheochromocytoma, renal cell carcinoma). The absence of tumor development in patients with congenital erythrocytosis remained unexplained. The R200W mutation is transmitted within a 340-kilobases haplotype inherited 14,000 to 62,000 years ago from a single founder event. The absence of tumor development in patients carrying the R200W mutation raised the possibility of the presence of a protective element within this core haplotype. Objective: The purpose of this study was to determine the mechanisms underlying the absence of tumor development in patients carrying the R200W mutation, healthy (heterozygous carriers) or with congenital erythrocytosis (homozygous). Methods: We report here one particular family carring a heterozygous R200W mutation associated, for the first time, with a typical von Hippel-Lindau disease (with pheochromocytoma, hemangioblastoma and renal cell carcinoma) (Olswang et al., 1998). We performed a comprehensive study with genotyping, phenotypic analysis, structural modeling, functional and transcriptomic studies of the R200W mutant in comparison to classical mutants involved in von Hippel-Lindau disease. For the first time, the use of inducible expression vectors encoding untagged VHL in the functional studies allowed the observation of very subtle effects. Results: We show that the R200W mutation alone is definitely not associated with the von Hippel Lindau disease because this particular family actually carries another pathogenic mutation, VHL-R161Q, on the same VHL allele, and the double mutated allele segregates with disease. Our genotyping showed that the doubled mutations lies on the Chuvash core haplotype transmitted with the R200W mutation. We can thus conclude that this haplotype does not contain any protective elements against cancer development and that the VHLmutations are, by themselves, predominantly responsible for manifestations of the von Hippel-Lindau disease. Functional studies demonstrate that the function of the R200W mutation is close to the wild type protein and the severity of disease manifestations observed in VHL syndrome is perfectly correlated with a gradient of pVHL dysfunction in hypoxia signaling pathways. Conclusion: We show that the R200W mutation associated with congenital erythrocytosis is at the bottom of an oncogenic gradient. These results validate for the first time in humans the recently proposed “continuum” model of tumor suppression, which differs from the classical Knudson’s two-hit model and implies that subtle gene dosage-sensitivity may underlie tumor predisposition. This model may be of major importance in our understanding of tumor risk in patients with erythrocytosis carrying germline mutations in other genes belonging the HIF pathway. Indeed, in rare cases, PHD2 and HIF2A pathogenic mutations also predispose patients to pheochromocytomas or paragangliomas development. Disclosures No relevant conflicts of interest to declare.

Development of V617F JAK2 Associated Myeloproliferative Neoplasms Is a Non-Random Event That Is Strongly Dependent on JAK2 Haplotype

Epidemiological data and family studies have indicated that inherited factors may predispose to the development of myeloproliferative neoplasms (MPN). It has also been suggested that single nucleotide polymorphisms (SNPs) within JAK2 are associated with specific MPN subtypes. To explore the role of inherited factors in more detail, we initially performed quantitative analysis of a series of JAK2 SNPs in homozygous PV cases (%V617F &gt;50%; n=73). Most mutant haplotypes could be read directly from the distorted allele ratios brought about by expansion of the homozygous clone. In many cases with 50–90% V617F, the residual wild type haplotype could also be read. Strikingly, of the 144 V617F alleles that could be determined, 111 (77%) had an identical core haplotype (subsequently designated 46/1) whereas only 9/76 (12%) residual wild type alleles were 46/1 (P = 1.9e-21, Fisher’s exact test). To explore this observation in more detail we first determined the haplotype structure of JAK2 using 14 SNPs genotyped by the Wellcome Trust Case Control Consortium (WTCCC) in 1500 UK blood donors. Nine haplotypes were inferred using the program PHASE that accounted for 94% of alleles, with a frequency of 0.24 for haplotype 46/1. Haplotype inference and tag SNP analysis revealed that 46/1 was also more frequent in heterozygous V617F positive MPD cases (135/354 alleles) compared to 188 locally sourced healthy controls (92/376 alleles; P = 0.0001) as well as the WTCCC cohort (P = 3.3e-8). Haplotype 46/1 was more frequent in all V617F positive disease entities compared to controls: PV (n=203; P=1.2e-16), ET (n=81; P=1.2e-9) and MF (n=41; P=8.0e-5) however there was no difference in the frequency of 46/1 between controls and V617F negative MPD / idiopathic erythrocytosis (n=123). To determine if heterozygous V617F also preferentially arose on a 46/1 allele as seen for homozygous cases, we developed an allele specific PCR between V617F and a SNP that tags this haplotype. In an analysis of 67 informative heterozygous V617F cases, 50 V617F alleles were 46/1 compared to only 17 residual wild type alleles (P=9.4e-9). We conclude that the 46/1 JAK2 haplotype is a strong predisposition factor for development of V617F associated MPDs (RR=2.6; 95% CI 2.3–2.9). The reason for this predisposition is currently unknown but it is likely that 46/1 is in linkage disequilibrium with an unknown constitutional functional variant that interacts with V617F JAK2.