Novel regulatory variant in ABO intronic RUNX1 binding site inducing A3 phenotype.

BACKGROUND AND OBJECTIVES: Mixed-field agglutination in ABO phenotyping (A3, B3) has been linked to genetically different blood cell populations such as in chimerism, or to rare variants in either ABO exon 7 or regulatory regions. Clarification of such cases is challenging and would greatly benefit from sequencing technologies that allow resolving full-gene haplotypes at high resolution. MATERIALS AND METHODS: We used long-read sequencing by Oxford Nanopore Technologies to sequence the entire ABO gene, amplified in two overlapping long-range PCR fragments, in a blood donor presented with A3B phenotype. Confirmation analyses were carried out by Sanger sequencing and included samples from other family members. RESULTS: Our data revealed a novel heterozygous g.10924C>A variant on the ABO*A allele located in the transcription factor binding site for RUNX1 in intron 1 (+5.8 kb site). Inheritance was shown by the results of the donor's mother, who shared the novel variant and the anti-A specific mixed-field agglutination. CONCLUSION: We discovered a regulatory variant in the 8-bp RUNX1 motif of ABO, which extends current knowledge of three other variants affecting the same motif and also leading to A3 or B3 phenotypes. Overall, long-range PCR combined with nanopore sequencing proved powerful and showed great potential as an emerging strategy for resolving cases with cryptic ABO phenotypes.

Two Prevalent ∼100-kb GYPB Deletions Causative of the GPB-Deficient Blood Group MNS Phenotype S-s-U- in Black Africans.

The U antigen (MNS5) is one of 49 antigens belonging to the MNS blood group system (ISBT002) carried on glycophorins A (GPA) and B (GPB). U is present on the red blood cells in almost all Europeans and Asians but absent in approximately 1.0% of Black Africans. U negativity coincides with negativity for S (MNS3) and s (MNS4) on GPB, thus be called S-s-U-, and is thought to arise from homozygous deletion of GYPB. Little is known about the molecular background of these deletions. Bioinformatic analysis of the 1000 Genomes Project data revealed several candidate regions with apparent deletions in GYPB. Highly specific Gap-PCRs, only resulting in positive amplification from DNAs with deletions present, allowed for the exact genetic localization of 3 different breakpoints; 110.24- and 103.26-kb deletions were proven to be the most frequent in Black Americans and Africans. Among 157 CEPH DNAs, deletions in 6 out of 8 African ethnicities were present. Allele frequencies of the deletions within African ethnicities varied greatly and reached a cumulative 23.3% among the Mbuti Pygmy people from the Congo. Similar observations were made for U+var alleles, known to cause strongly reduced GPB expression. The 110- and 103-kb deletional GYPB haplotypes were found to represent the most prevalent hereditary factors causative of the MNS blood group phenotype S-s-U-. Respective GYPB deletions are now accessible by molecular detection of homo- and hemizygous transmission.

Tandem repeat variation in human and great ape populations and its impact on gene expression divergence.

Tandem repeats (TRs) are stretches of DNA that are highly variable in length and mutate rapidly. They are thus an important source of genetic variation. This variation is highly informative for population and conservation genetics. It has also been associated with several pathological conditions and with gene expression regulation. However, genome-wide surveys of TR variation in humans and closely related species have been scarce due to technical difficulties derived from short-read technology. Here we explored the genome-wide diversity of TRs in a panel of 83 human and nonhuman great ape genomes, in a total of six different species, and studied their impact on gene expression evolution. We found that population diversity patterns can be efficiently captured with short TRs (repeat unit length, 1-5 bp). We examined the potential evolutionary role of TRs in gene expression differences between humans and primates by using 30,275 larger TRs (repeat unit length, 2-50 bp). Genes that contained TRs in the promoters, in their 3' untranslated region, in introns, and in exons had higher expression divergence than genes without repeats in the regions. Polymorphic small repeats (1-5 bp) had also higher expression divergence compared with genes with fixed or no TRs in the gene promoters. Our findings highlight the potential contribution of TRs to human evolution through gene regulation.

Stepwise partitioning of Xp21: a profiling method for XK deletions causative of the McLeod syndrome.

BACKGROUND: McLeod syndrome (MLS) is hematologically defined by the absence of the red blood cell (RBC) antigen Kx on the transmembrane RBC protein, XK, representing a highly specific diagnostic marker. Direct molecular assessment of XK therefore represents a desirable diagnostic tool. Whereas pathogenic point mutations may be simply identified, partial and complete deletions of XK on Xp21.1, eventually covering adjacent genes and causing multifaceted "continuous gene syndromes," are difficult to localize. STUDY DESIGN AND METHODS: Three different McLeod patient samples were tested using 16 initial positional polymerase chain reaction (PCR) procedures distributed over an approximately 2.8-Mbp Xp-chromosomal region, ranging telomeric from MAGEB16 to OTC, centromeric of XK. The molecular breakpoint of one sample with an apparent large Xp deletion was iteratively narrowed down by stepwise positioning further PCR procedures and sequenced. Two mutant XK genes, one previously published and serving as a positive control, were also sequenced. RESULTS: We confirmed the positive control as previously published and listed as XK*N.20 by the International Society of Blood Transfusion (ISBT). The other XK showed a novel four-nucleotide deletion in Exon 1, 195-198delCCGC (newly listed as XK*N.39 by the ISBT). The third sample had an approximately 151-kbp X-chromosomal deletion, reaching from Exon 2 of LANCL3, across XK to Exon 3 of CYBB (newly listed as XK*N.01.016 by the ISBT). Carrier status of the patients' sister was diagnosed using a diagnostic "gap-PCR." CONCLUSIONS: The stepwise partitioning of Xp21.1 is pragmatic and cost-efficient in comparison to other diagnostic techniques such as "massive parallel sequencing" given the rarity of MLS. All males with suspected MLS should be considered for molecular XK profiling.

Reconstructing the demographic history of orang-utans using Approximate Bayesian Computation.

Investigating how different evolutionary forces have shaped patterns of DNA variation within and among species requires detailed knowledge of their demographic history. Orang-utans, whose distribution is currently restricted to the South-East Asian islands of Borneo (Pongo pygmaeus) and Sumatra (Pongo abelii), have likely experienced a complex demographic history, influenced by recurrent changes in climate and sea levels, volcanic activities and anthropogenic pressures. Using the most extensive sample set of wild orang-utans to date, we employed an Approximate Bayesian Computation (ABC) approach to test the fit of 12 different demographic scenarios to the observed patterns of variation in autosomal, X-chromosomal, mitochondrial and Y-chromosomal markers. In the best-fitting model, Sumatran orang-utans exhibit a deep split of populations north and south of Lake Toba, probably caused by multiple eruptions of the Toba volcano. In addition, we found signals for a strong decline in all Sumatran populations ~24 ka, probably associated with hunting by human colonizers. In contrast, Bornean orang-utans experienced a severe bottleneck ~135 ka, followed by a population expansion and substructuring starting ~82 ka, which we link to an expansion from a glacial refugium. We showed that orang-utans went through drastic changes in population size and connectedness, caused by recurrent contraction and expansion of rainforest habitat during Pleistocene glaciations and probably hunting by early humans. Our findings emphasize the fact that important aspects of the evolutionary past of species with complex demographic histories might remain obscured when applying overly simplified models.

Generation of SNP datasets for orangutan population genomics using improved reduced-representation sequencing and direct comparisons of SNP calling algorithms.

BACKGROUND: High-throughput sequencing has opened up exciting possibilities in population and conservation genetics by enabling the assessment of genetic variation at genome-wide scales. One approach to reduce genome complexity, i.e. investigating only parts of the genome, is reduced-representation library (RRL) sequencing. Like similar approaches, RRL sequencing reduces ascertainment bias due to simultaneous discovery and genotyping of single-nucleotide polymorphisms (SNPs) and does not require reference genomes. Yet, generating such datasets remains challenging due to laboratory and bioinformatical issues. In the laboratory, current protocols require improvements with regards to sequencing homologous fragments to reduce the number of missing genotypes. From the bioinformatical perspective, the reliance of most studies on a single SNP caller disregards the possibility that different algorithms may produce disparate SNP datasets. RESULTS: We present an improved RRL (iRRL) protocol that maximizes the generation of homologous DNA sequences, thus achieving improved genotyping-by-sequencing efficiency. Our modifications facilitate generation of single-sample libraries, enabling individual genotype assignments instead of pooled-sample analysis. We sequenced ~1% of the orangutan genome with 41-fold median coverage in 31 wild-born individuals from two populations. SNPs and genotypes were called using three different algorithms. We obtained substantially different SNP datasets depending on the SNP caller. Genotype validations revealed that the Unified Genotyper of the Genome Analysis Toolkit and SAMtools performed significantly better than a caller from CLC Genomics Workbench (CLC). Of all conflicting genotype calls, CLC was only correct in 17% of the cases. Furthermore, conflicting genotypes between two algorithms showed a systematic bias in that one caller almost exclusively assigned heterozygotes, while the other one almost exclusively assigned homozygotes. CONCLUSIONS: Our enhanced iRRL approach greatly facilitates genotyping-by-sequencing and thus direct estimates of allele frequencies. Our direct comparison of three commonly used SNP callers emphasizes the need to question the accuracy of SNP and genotype calling, as we obtained considerably different SNP datasets depending on caller algorithms, sequencing depths and filtering criteria. These differences affected scans for signatures of natural selection, but will also exert undue influences on demographic inferences. This study presents the first effort to generate a population genomic dataset for wild-born orangutans with known population provenance.

Resolving Genotype-Phenotype Discrepancies of the Kidd Blood Group System Using Long-Read Nanopore Sequencing.

Due to substantial improvements in read accuracy, third-generation long-read sequencing holds great potential in blood group diagnostics, particularly in cases where traditional genotyping or sequencing techniques, primarily targeting exons, fail to explain serological phenotypes. In this study, we employed Oxford Nanopore sequencing to resolve all genotype-phenotype discrepancies in the Kidd blood group system (JK, encoded by SLC14A1) observed over seven years of routine high-throughput donor genotyping using a mass spectrometry-based platform at the Blood Transfusion Service, Zurich. Discrepant results from standard serological typing and donor genotyping were confirmed using commercial PCR-SSP kits. To resolve discrepancies, we amplified the entire coding region of SLC14A1 (~24 kb, exons 3 to 10) in two overlapping long-range PCRs in all samples. Amplicons were barcoded and sequenced on a MinION flow cell. Sanger sequencing and bridge-PCRs were used to confirm findings. Among 11,972 donors with both serological and genotype data available for the Kidd system, we identified 10 cases with unexplained conflicting results. Five were linked to known weak and null alleles caused by variants not included in the routine donor genotyping. In two cases, we identified novel null alleles on the JK*01 (Gly40Asp; c.119G>A) and JK*02 (Gly242Glu; c.725G>A) haplotypes, respectively. Remarkably, the remaining three cases were associated with a yet unknown deletion of ~5 kb spanning exons 9-10 of the JK*01 allele, which other molecular methods had failed to detect. Overall, nanopore sequencing demonstrated reliable and accurate performance for detecting both single-nucleotide and structural variants. It possesses the potential to become a robust tool in the molecular diagnostic portfolio, particularly for addressing challenging structural variants such as hybrid genes, deletions and duplications.

Marked population structure and recent migration in the critically endangered Sumatran orangutan (Pongo abelii).

A multitude of factors influence how natural populations are genetically structured, including dispersal barriers, inhomogeneous habitats, and social organization. Such population subdivision is of special concern in endangered species, as it may lead to reduced adaptive potential and inbreeding in local subpopulations, thus increasing the risk of future extinctions. With only 6600 animals left in the wild, Sumatran orangutans (Pongo abelii) are among the most endangered, but also most enigmatic, great ape species. In order to infer the fine-scale population structure and connectivity of Sumatran orangutans, we analyzed the most comprehensive set of samples to date, including mitochondrial hyper-variable region I haplotypes for 123 individuals and genotypes of 27 autosomal microsatellite markers for 109 individuals. For both mitochondrial and autosomal markers, we found a pronounced population structure, caused by major rivers, mountain ridges, and the Toba caldera. We found that genetic diversity and corresponding long-term effective population size estimates vary strongly among sampling regions for mitochondrial DNA, but show remarkable similarity for autosomal markers, hinting at male-driven long-distance gene flow. In support of this, we identified several individuals that were most likely sired by males originating from other genetic clusters. Our results highlight the effect of natural barriers in shaping the genetic structure of great ape populations, but also point toward important dispersal corridors on northern Sumatra that allow for genetic exchange.

First Report of a Low-Frequency Mosaic Mutation in the Hydroxymethylbilane Synthase Gene Causing Acute Intermittent Porphyria.

Acute porphyrias are a group of monogenetic inborn errors of heme biosynthesis, characterized by acute and potentially life-threatening neurovisceral attacks upon exposure to certain triggering factors. Biochemical analyses can determine the type of acute porphyria, and subsequent genetic analysis allows for the identification of pathogenic variants in the specific gene, which provides information for family counselling. In 2017, a male Swiss patient was diagnosed with an acute porphyria while suffering from an acute attack. The pattern of porphyrin metabolite excretion in urine, faeces, and plasma was typical for an acute intermittent porphyria (AIP), which is caused by inherited autosomal dominant mutations in the gene for hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. However, the measurement of HMBS enzymatic activity in the erythrocytes was within the normal range and Sanger sequencing of the HMBS gene failed to detect any pathogenic variants. To explore the molecular basis of the apparent AIP in this patient, we performed third-generation long-read single-molecule sequencing (nanopore sequencing) on a PCR product spanning the entire HMBS gene, including the intronic sequences. We identified a known pathogenic variant, c.77G>A, p.(Arg26His), in exon 3 at an allelic frequency of ~22% in the patient's blood. The absence of the pathogenic variant in the DNA of the parents and the results of additional confirmatory studies supported the presence of a de novo mosaic mutation. To our knowledge, such a mutation has not been previously described in any acute porphyria. Therefore, de novo mosaic mutations should be considered as potential causes of acute porphyrias when no pathogenic genetic variant can be identified through routine molecular diagnostics.

Haplotype sequence collection of ABO blood group alleles by long-read sequencing reveals putative A1-diagnostic variants.

In the era of blood group genomics, reference collections of complete and fully resolved blood group gene alleles have gained high importance. For most blood groups, however, such collections are currently lacking, as resolving full-length gene sequences as haplotypes (ie, separated maternal/paternal origin) remains exceedingly difficult with both Sanger and short-read next-generation sequencing. Using the latest third-generation long-read sequencing, we generated a collection of fully resolved sequences for all 6 main ABO allele groups: ABO∗A1/A2/B/O.01.01/O.01.02/O.02. We selected 77 samples from an ABO genotype data set (n = 25 200) of serologically typed Swiss blood donors. The entire ABO gene was amplified in 2 overlapping long-range polymerase chain reactions (covering ∼23.6 kb) and sequenced by long-read Oxford Nanopore sequencing. For quality validation, 2 samples per ABO group were resequenced using Illumina and Pacific Biosciences technology. All 154 full-length ABO sequences were resolved as haplotypes. We observed novel, distinct sequence patterns for each ABO group. Most genetic diversity was found between, not within, ABO groups. Phylogenetic tree and haplotype network analyses highlighted distinct clades of each ABO group. Strikingly, our data uncovered 4 genetic variants putatively specific for ABO∗A1, for which direct diagnostic targets are currently lacking. We validated A1-diagnostic potential using whole-genome data (n = 4872) of a multiethnic cohort. Overall, our sequencing strategy proved powerful for producing high-quality ABO haplotypes and holds promise for generating similar collections for other blood groups. The publicly available collection of 154 haplotypes will serve as a valuable resource for molecular analyses of ABO, as well as studies about the function and evolutionary history of ABO.

Genomes reveal marked differences in the adaptive evolution between orangutan species.

BACKGROUND: Integrating demography and adaptive evolution is pivotal to understanding the evolutionary history and conservation of great apes. However, little is known about the adaptive evolution of our closest relatives, in particular if and to what extent adaptions to environmental differences have occurred. Here, we used whole-genome sequencing data from critically endangered orangutans from North Sumatra (Pongo abelii) and Borneo (P. pygmaeus) to investigate adaptive responses of each species to environmental differences during the Pleistocene. RESULTS: Taking into account the markedly disparate demographic histories of each species after their split ~ 1 Ma ago, we show that persistent environmental differences on each island had a strong impact on the adaptive evolution of the genus Pongo. Across a range of tests for positive selection, we find a consistent pattern of between-island and species differences. In the more productive Sumatran environment, the most notable signals of positive selection involve genes linked to brain and neuronal development, learning, and glucose metabolism. On Borneo, however, positive selection comprised genes involved in lipid metabolism, as well as cardiac and muscle activities. CONCLUSIONS: We find strikingly different sets of genes appearing to have evolved under strong positive selection in each species. In Sumatran orangutans, selection patterns were congruent with well-documented cognitive and behavioral differences between the species, such as a larger and more complex cultural repertoire and higher degrees of sociality. However, in Bornean orangutans, selective responses to fluctuating environmental conditions appear to have produced physiological adaptations to generally lower and temporally more unpredictable food supplies.

Morphometric, Behavioral, and Genomic Evidence for a New Orangutan Species.

Six extant species of non-human great apes are currently recognized: Sumatran and Bornean orangutans, eastern and western gorillas, and chimpanzees and bonobos [1]. However, large gaps remain in our knowledge of fine-scale variation in hominoid morphology, behavior, and genetics, and aspects of great ape taxonomy remain in flux. This is particularly true for orangutans (genus: Pongo), the only Asian great apes and phylogenetically our most distant relatives among extant hominids [1]. Designation of Bornean and Sumatran orangutans, P. pygmaeus (Linnaeus 1760) and P. abelii (Lesson 1827), as distinct species occurred in 2001 [1, 2]. Here, we show that an isolated population from Batang Toru, at the southernmost range limit of extant Sumatran orangutans south of Lake Toba, is distinct from other northern Sumatran and Bornean populations. By comparing cranio-mandibular and dental characters of an orangutan killed in a human-animal conflict to those of 33 adult male orangutans of a similar developmental stage, we found consistent differences between the Batang Toru individual and other extant Ponginae. Our analyses of 37 orangutan genomes provided a second line of evidence. Model-based approaches revealed that the deepest split in the evolutionary history of extant orangutans occurred ∼3.38 mya between the Batang Toru population and those to the north of Lake Toba, whereas both currently recognized species separated much later, about 674 kya. Our combined analyses support a new classification of orangutans into three extant species. The new species, Pongo tapanuliensis, encompasses the Batang Toru population, of which fewer than 800 individuals survive. VIDEO ABSTRACT.

Evolution and demography of the great apes.

The great apes are the closest living relatives of humans. Chimpanzees and bonobos group together with humans, while gorillas and orangutans are more divergent from humans. Here, we review insights into their evolution pertaining to the topology of species and subspecies and the reconstruction of their demography based on genome-wide variation. These advances have only become possible recently through next-generation sequencing technologies. Given the close relationship to humans, they provide an important evolutionary context for human genetics.

The quest for Y-chromosomal markers - methodological strategies for mammalian non-model organisms.

Tracing maternal and paternal lineages independently to explore breeding systems and dispersal strategies in natural populations has been high on the wish-list of evolutionary biologists. As males are the heterogametic sex in mammals, such sex-specific patterns can be indirectly observed when Y chromosome polymorphism is combined with mitochondrial sequence information. Over the past decade, Y-chromosomal markers applied to human populations have revealed remarkable differences in the demographic history and behaviour between the sexes. However, with a few exceptions, genetic data tracing the paternal line are lacking in most other mammalian species. This deficit can be attributed to the difficulty of developing Y-specific genetic markers in non-model organisms and the general low levels of polymorphisms observed on the Y chromosome. Here, we present an overview of the currently employed strategies for developing paternal markers in mammals. Moreover, we review the practical feasibility and requirements of various methodological strategies and highlight their future prospects when combined with new molecular techniques such as next generation sequencing.

Development of polymorphic microsatellite markers for the dung fly (Sepsis cynipsea).

The polyandrous fly Sepsis cynipsea has been used extensively in studies of sexual selection and local adaptation. We isolated and characterized 11 novel microsatellite markers for S. cynipsea from a genomic library and screened 32 flies for polymorphism. All microsatellite markers show high allelic diversity with an average of 9.64 alleles per locus. Two microsatellites were found likely to be X-linked. These novel markers will significantly advance studies of sexual selection and evolutionary genetics of S. cynipsea and related species, especially given the low numbers of markers currently available in this family.