Genomic analyses of hair from Ludwig van Beethoven.

Ludwig van Beethoven (1770-1827) remains among the most influential and popular classical music composers. Health problems significantly impacted his career as a composer and pianist, including progressive hearing loss, recurring gastrointestinal complaints, and liver disease. In 1802, Beethoven requested that following his death, his disease be described and made public. Medical biographers have since proposed numerous hypotheses, including many substantially heritable conditions. Here we attempt a genomic analysis of Beethoven in order to elucidate potential underlying genetic and infectious causes of his illnesses. We incorporated improvements in ancient DNA methods into existing protocols for ancient hair samples, enabling the sequencing of high-coverage genomes from small quantities of historical hair. We analyzed eight independently sourced locks of hair attributed to Beethoven, five of which originated from a single European male. We deemed these matching samples to be almost certainly authentic and sequenced Beethoven's genome to 24-fold genomic coverage. Although we could not identify a genetic explanation for Beethoven's hearing disorder or gastrointestinal problems, we found that Beethoven had a genetic predisposition for liver disease. Metagenomic analyses revealed furthermore that Beethoven had a hepatitis B infection during at least the months prior to his death. Together with the genetic predisposition and his broadly accepted alcohol consumption, these present plausible explanations for Beethoven's severe liver disease, which culminated in his death. Unexpectedly, an analysis of Y chromosomes sequenced from five living members of the Van Beethoven patrilineage revealed the occurrence of an extra-pair paternity event in Ludwig van Beethoven's patrilineal ancestry.

Ancestral patterns of recessive dystrophic epidermolysis bullosa mutations in Hispanic populations suggest sephardic ancestry.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genodermatosis caused by mutations in the gene coding for type VII collagen (COL7A1). More than 800 different pathogenic mutations in COL7A1 have been described to date; however, the ancestral origins of many of these mutations have not been precisely identified. In this study, 32 RDEB patient samples from the Southwestern United States, Mexico, Chile, and Colombia carrying common mutations in the COL7A1 gene were investigated to determine the origins of these mutations and the extent to which shared ancestry contributes to disease prevalence. The results demonstrate both shared European and American origins of RDEB mutations in distinct populations in the Americas and suggest the influence of Sephardic ancestry in at least some RDEB mutations of European origins. Knowledge of ancestry and relatedness among RDEB patient populations will be crucial for the development of future clinical trials and the advancement of novel therapeutics.

Phylogenetic history of patrilineages rare in northern and eastern Europe from large-scale re-sequencing of human Y-chromosomes.

The most frequent Y-chromosomal (chrY) haplogroups in northern and eastern Europe (NEE) are well-known and thoroughly characterised. Yet a considerable number of men in every population carry rare paternal lineages with estimated frequencies around 5%. So far, limited sample-sizes and insufficient resolution of genotyping have obstructed a truly comprehensive look into the variety of rare paternal lineages segregating within populations and potential signals of population history that such lineages might convey. Here we harness the power of massive re-sequencing of human Y chromosomes to identify previously unknown population-specific clusters among rare paternal lineages in NEE. We construct dated phylogenies for haplogroups E2-M215, J2-M172, G-M201 and Q-M242 on the basis of 421 (of them 282 novel) high-coverage chrY sequences collected from large-scale databases focusing on populations of NEE. Within these otherwise rare haplogroups we disclose lineages that began to radiate ~1-3 thousand years ago in Estonia and Sweden and reveal male phylogenetic patterns testifying of comparatively recent local demographic expansions. Conversely, haplogroup Q lineages bear evidence of ancient Siberian influence lingering in the modern paternal gene pool of northern Europe. We assess the possible direction of influx of ancestral carriers for some of these male lineages. In addition, we demonstrate the congruency of paternal haplogroup composition of our dataset with two independent population-based cohorts from Estonia and Sweden.

Origin and diffusion of human Y chromosome haplogroup J1-M267.

Human Y chromosome haplogroup J1-M267 is a common male lineage in West Asia. One high-frequency region-encompassing the Arabian Peninsula, southern Mesopotamia, and the southern Levant-resides ~ 2000 km away from the other one found in the Caucasus. The region between them, although has a lower frequency, nevertheless demonstrates high genetic diversity. Studies associate this haplogroup with the spread of farming from the Fertile Crescent to Europe, the spread of mobile pastoralism in the desert regions of the Arabian Peninsula, the history of the Jews, and the spread of Islam. Here, we study past human male demography in West Asia with 172 high-coverage whole Y chromosome sequences and 889 genotyped samples of haplogroup J1-M267. We show that this haplogroup evolved ~ 20,000 years ago somewhere in northwestern Iran, the Caucasus, the Armenian Highland, and northern Mesopotamia. The major branch-J1a1a1-P58-evolved during the early Holocene ~ 9500 years ago somewhere in the Arabian Peninsula, the Levant, and southern Mesopotamia. Haplogroup J1-M267 expanded during the Chalcolithic, the Bronze Age, and the Iron Age. Most probably, the spread of Afro-Asiatic languages, the spread of mobile pastoralism in the arid zones, or both of these events together explain the distribution of haplogroup J1-M267 we see today in the southern regions of West Asia.

Performance comparison: exome sequencing as a single test replacing Sanger sequencing.

Next generation sequencing tests are used routinely as first-choice tests in the clinic. However, systematic performance comparing the results of exome sequencing as a single test replacing Sanger sequencing of targeted gene(s) is still lacking. Performance comparison data are critically important for clinical case management. In this study, we compared Sanger-sequencing results of 258 genes to those obtained from next generation sequencing (NGS) using two exome-sequencing enrichment kits: Agilent-SureSelectQXT and Illumina-Nextera. Sequencing was performed on leukocytes and buccal-derived DNA from a single individual, and all 258 genes were sequenced a total of 11 times (using different sequencing methods and DNA sources). Sanger sequencing was completed for all exons, including flanking ± 8 bp regions. For the 258 genes, NGS mean coverage was > 20 × for > 98 and > 91% of the regions targeted by SureSelect and Nextera, respectively. Overall, 449 variants were identified in at least one experiment, and 407/449 (90.6%) were detected by all. Of the 42 discordant variants, 23 were determined as true calls, summing-up to a truth set of 430 variants. Sensitivity of true-variant detection was 99% for Sanger sequencing and 97-100% for the NGS experiments. Mean false-positive rates were 3.7E-6 for Sanger sequencing, 2.5E-6 for SureSelect-NGS and 5.2E-6 for Nextera-NGS. Our findings suggest a high overall concordance between Sanger sequencing and NGS performances. Both methods demonstrated false-positive and false-negative calls. High clinical suspicion for a specific diagnosis should, therefore, override negative results of either Sanger sequencing or NGS.

Dissecting the paternal founders of Mundari (Austroasiatic) speakers associated with the language dispersal in South Asia.

The phylogenetic analysis of Y chromosomal haplogroup O2a-M95 was crucial to determine the nested structure of South Asian branches within the larger tree, predominantly present in East and Southeast Asia. However, it had previously been unclear that how many founders brought the haplogroup O2a-M95 to South Asia. On the basis of the updated Y chromosomal tree for haplogroup O2a-M95, we analysed 1437 male samples from South Asia for various novel downstream markers, carefully selected from the extant phylogenetic tree. With this increased resolution of genetic markers, we were able to identify at least three founders downstream to haplogroup O2a-M95, who are likely to have been associated with the dispersal of Austroasiatic languages to South Asia. The fourth founder was exclusively present amongst Tibeto-Burman speakers of Manipur and Bangladesh. In sum, our new results suggest the arrival of Austroasiatic languages in South Asia during last 5000 years.

Determination of the phylogenetic origins of the Árpád Dynasty based on Y chromosome sequencing of Béla the Third.

We set out to identify the origins of the Árpád Dynasty based on genome sequencing of DNA derived from the skeletal remains of Hungarian King Béla III (1172-1196) and eight additional individuals (six males, two females) originally interred at the Royal Basilica of Székesfehérvár. Y-chromosome analysis established that two individuals, Béla III and HU52 assign to haplogroups R-Z2125 whose distribution centres near South Central Asia with subsidiary expansions in the regions of modern Iran, the Volga Ural region and the Caucasus. Out of a cohort of 4340 individuals from these geographic areas, we acquired whole-genome data from 208 individuals derived for the R-Z2123 haplogroup. From these data we have established that the closest living kin of the Árpád Dynasty are R-SUR51 derived modern day Bashkirs predominantly from the Burzyansky and Abzelilovsky districts of Bashkortostan in the Russian Federation. Our analysis also reveals the existence of SNPs defining a novel Árpád Dynasty specific haplogroup R-ARP. Framed within the context of a high resolution R-Z2123 phylogeny, the ancestry of the first Hungarian royal dynasty traces to the region centering near Northern Afghanistan about 4500 years ago and identifies the Bashkirs as their closest kin, with a separation date between the two populations at the beginning of the first millennium CE.

Teaching clinicians practical genomic medicine: 7 years' experience in a tertiary care center.

PURPOSE: Increased implementation of complex genetic technologies in clinical practice emphasizes the urgency of genomic literacy and proficiency for medical professionals. We evaluated our genomic education model. METHODS: We assessed the 5-day, extended format program, encompassing lectures, videos, interactive tests, practice cases, and clinical exercises. Pre- and post questionnaires assessed knowledge change, using t-tests to compare groups. Satisfaction on program completion and after 3 years were evaluated. Implementation in other centers determined acceptability. RESULTS: During 2012-2018, 774 clinicians from multiple disciplines and career stages attended 35 programs; 334 (43%) attended the 5-day extended format. Evaluations showed significant improvement of genomic literacy (mean 15.05/100 points, p < 0.001). Residents initially had higher scores than specialists (pre: 66.3 ± 17.3 vs. 58.7 ± 16.6, respectively, p = 0.002); both significantly improved, with specialists "catching up" (post: 79.1 ± 17.2 vs. 75.7 ± 15.9, nonsignificant (NS)); there was a similar trend between fellows and subspecialists (pre: 70 ± 18 vs. 59.4 ± 16.4, respectively, p = 0.007; post: 78.6 ± 16.4 vs. 73.2 ± 17.7, respectively, NS). Younger specialists (≤10 years residency) had significantly higher pre- and post scores. Absolute improvement in scores did not depend on medical specialties. CONCLUSION: Our program is effective in improving genomics literacy for clinicians, irrespective of career length or expertise, and could be a model for improving skills in practical genomics for all medical professionals.

The novel founder homozygous V225M mutation in the HSD17B3 gene causes aberrant splicing and XY-DSD.

PURPOSE: Mutations in the gene HSD17B3 encoding the 17-beta hydroxysteroid dehydrogenase 3 enzyme cause testosterone insufficiency leading to XY disorders of sex development. In this study the clinical and molecular characteristics of three patients from consanguineous families are elucidated. METHODS: We identified three patients from two unrelated families with XY DSD and a novel homozygous HSD17B3:c. 673G>A mutation. The effect of the mutation on splicing was determined in RNA extracted from the testis of one patient. RESULTS: Three patients presented at ages 0.1, 8 and 0.7 years with ambiguous genitalia and an XY Karyotype. Endocrine workup showed normal cortisol and mineralocorticoid levels with a low testosterone/androstenedione ratio. Whole-exome sequencing, carried out in the first family, revealed a homozygous novel mutation in the HSD17B3 gene: c. 673G>A, p. V225M. The same mutation was found by Sanger sequencing in the third unrelated patient. Haplotype analysis of a 4 Mb region surrounding the HSD17B3 gene on chromosome 9 revealed that the mutation resides on the same allele in all three patients. The mutation, being the first nucleic acid on exon 10, affects splicing and causes exon 10 skipping in one of our patients' testes. CONCLUSION: The novel homozygous c. 673G>A, p. V225M mutation in the 17HSDB3 gene is likely a founder mutation and causes severe XY-DSD. It changes a conserved amino acid residue, and also alters 17HSDB3 gene transcription by causing skipping of exon 10, thereby contributing to an imbalance in the relevant protein isoforms and consequently, significant decreased 17HDSB3 enzymatic activity.

Preconception carrier screening yield: effect of variants of unknown significance in partners of carriers with clinically significant variants.

PURPOSE: Expanded preconception carrier screening (ECS) identifies at-risk couples (ARCs) for multiple diseases. ECS reports currently include only pathogenic/likely pathogenic variants (P/LPVs). Variants of unknown significance (VUS) are not reported, unlike genomic or chromosomal array test results in other post/prenatal settings. Couples who are P/LP and VUS carriers (P/LP*VUS) may be at risk, particularly in genes with high P/LP carrier rates. We examined the possible contribution of P/LP*cVUS (coding, nonsynonymous VUS) matings to ECS yield in an Ashkenazi Jewish cohort, a population with well-established preconception screening. METHODS: We analyzed 672 Ashkenazi Jewish genome sequences (225,456 virtual matings) for variants in three different gene sets and calculated the rates of P/LP*P/LP and P/LP*cVUS matings. RESULTS: Across 180 genes, we identified 4671 variants: 144 (3.1%) P/LP and 1963 (42%) VUS. Across gene sets, the proportion of P/LP*P/LP and P/LP*cVUS ARCs was 2.7-3.8% and 6.8-7.5%, respectively. CONCLUSION: Disregarding VUS in ECS may miss ARCs. Even if only 10% of couples currently classified as P/LP*cVUS are ultimately reclassified as P/LP*P/LP, ECS yield would increase by ≈20%. While current understanding of VUS precludes VUS reporting in ECS, these findings underscore the importance of VUS reclassification. This will crucially depend on enlarging population frequency databases, especially of affected individuals.

West Asian sources of the Eurasian component in Ethiopians: a reassessment.

The presence of genomic signatures of Eurasian origin in contemporary Ethiopians has been reported by several authors and estimated to have arrived in the area from 3000 years ago. Several studies reported plausible source populations for such a signature, using haplotype based methods on modern data or single-site methods on modern or ancient data. These studies did not reach a consensus and suggested an Anatolian or Sardinia-like proxy, broadly Levantine or Neolithic Levantine as possible sources. We demonstrate, however, that the deeply divergent, autochthonous African component which accounts for ~50% of most contemporary Ethiopian genomes, affects the overall allele frequency spectrum to an extent that makes it hard to control for it and, at once, to discern between subtly different, yet important, Eurasian sources (such as Anatolian or Levant Neolithic ones). Here we re-assess pattern of allele sharing between the Eurasian component of Ethiopians (here called "NAF" for Non African) and ancient and modern proxies. Our results unveil a genomic legacy that may connect the Eurasian genetic component of contemporary Ethiopians with Sea People and with population movements that affected the Mediterranean area and the Levant after the fall of the Minoan civilization.

The spectrum of BRCA1 and BRCA2 pathogenic sequence variants in Middle Eastern, North African, and South European countries.

BRCA1 BRCA2 mutational spectrum in the Middle East, North Africa, and Southern Europe is not well characterized. The unique history and cultural practices characterizing these regions, often involving consanguinity and inbreeding, plausibly led to the accumulation of population-specific founder pathogenic sequence variants (PSVs). To determine recurring BRCA PSVs in these locales, a search in PUBMED, EMBASE, BIC, and CIMBA was carried out combined with outreach to researchers from the relevant countries for unpublished data. We identified 232 PSVs in BRCA1 and 239 in BRCA2 in 25 of 33 countries surveyed. Common PSVs that were detected in four or more countries were c.5266dup (p.Gln1756Profs), c.181T>G (p.Cys61Gly), c.68_69del (p.Glu23Valfs), c.5030_5033del (p.Thr1677Ilefs), c.4327C>T (p.Arg1443Ter), c.5251C>T (p.Arg1751Ter), c.1016dup (p.Val340Glyfs), c.3700_3704del (p.Val1234Glnfs), c.4065_4068del (p.Asn1355Lysfs), c.1504_1508del (p.Leu502Alafs), c.843_846del (p.Ser282Tyrfs), c.798_799del (p.Ser267Lysfs), and c.3607C>T (p.Arg1203Ter) in BRCA1 and c.2808_2811del (p.Ala938Profs), c.5722_5723del (p.Leu1908Argfs), c.9097dup (p.Thr3033Asnfs), c.1310_1313del (p. p.Lys437Ilefs), and c.5946del (p.Ser1982Argfs) for BRCA2. Notably, some mutations (e.g., p.Asn257Lysfs (c.771_775del)) were observed in unrelated populations. Thus, seemingly genotyping recurring BRCA PSVs in specific populations may provide first pass BRCA genotyping platform.

Author Correction: The genetic legacy of continental scale admixture in Indian Austroasiatic speakers.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Influence of genetic copy number variants of the human GLUT3 glucose transporter gene SLC2A3 on protein expression, glycolysis and rheumatoid arthritis risk: A genetic replication study.

OBJECTIVES: The gene encoding glucose transporter 3 (GLUT3, SLC2A3) is present in the human population at variable copy number. An overt disease phenotype of SLC2A3 copy number variants has not been reported; however, deletion of SLC2A3 has been previously reported to protect carriers from rheumatoid arthritis, implicating GLUT3 as a therapeutic target in rheumatoid arthritis. Here we aim to perform functional analysis of GLUT3 copy number variants in immune cells, and test the reported protective association of the GLUT3 copy number variants for rheumatoid arthritis in a genetic replication study. METHODS: Cells from genotyped healthy controls were analyzed for SLC2A3/GLUT3 expression and glycolysis capacity. We genotyped the SLC2A3 copy number variant in four independent cohorts of rheumatoid arthritis and controls and one cohort of multiple sclerosis and controls. RESULTS: Heterozygous deletion of SLC2A3 correlates directly with expression levels of GLUT3 and influences glycolysis rates in the human immune system. The frequency of the SLC2A3 copy number variant is not different between rheumatoid arthritis, multiple sclerosis and control groups. CONCLUSIONS: Despite a robust SLC2A3 gene copy number dependent phenotype, our study of large groups of rheumatoid arthritis cases and controls provides no evidence for rheumatoid arthritis disease protection in deletion carriers. These data emphasize the importance of well powered replication studies to confirm or refute genetic associations, particularly for relatively rare variants.

The genetic legacy of continental scale admixture in Indian Austroasiatic speakers.

Surrounded by speakers of Indo-European, Dravidian and Tibeto-Burman languages, around 11 million Munda (a branch of Austroasiatic language family) speakers live in the densely populated and genetically diverse South Asia. Their genetic makeup holds components characteristic of South Asians as well as Southeast Asians. The admixture time between these components has been previously estimated on the basis of archaeology, linguistics and uniparental markers. Using genome-wide genotype data of 102 Munda speakers and contextual data from South and Southeast Asia, we retrieved admixture dates between 2000-3800 years ago for different populations of Munda. The best modern proxies for the source populations for the admixture with proportions 0.29/0.71 are Lao people from Laos and Dravidian speakers from Kerala in India. The South Asian population(s), with whom the incoming Southeast Asians intermixed, had a smaller proportion of West Eurasian genetic component than contemporary proxies. Somewhat surprisingly Malaysian Peninsular tribes rather than the geographically closer Austroasiatic languages speakers like Vietnamese and Cambodians show highest sharing of IBD segments with the Munda. In addition, we affirmed that the grouping of the Munda speakers into North and South Munda based on linguistics is in concordance with genome-wide data.

Reconstructing the demographic history of the Himalayan and adjoining populations.

The rugged topography of the Himalayan region has hindered large-scale human migrations, population admixture and assimilation. Such complexity in geographical structure might have facilitated the existence of several small isolated communities in this region. We have genotyped about 850,000 autosomal markers among 35 individuals belonging to the four major populations inhabiting the Himalaya and adjoining regions. In addition, we have genotyped 794 individuals belonging to 16 ethnic groups from the same region, for uniparental (mitochondrial and Y chromosomal DNA) markers. Our results in the light of various statistical analyses suggest a closer link of the Himalayan and adjoining populations to East Asia than their immediate geographical neighbours in South Asia. Allele frequency-based analyses likely support the existence of a specific ancestry component in the Himalayan and adjoining populations. The admixture time estimate suggests a recent westward migration of populations living to the East of the Himalaya. Furthermore, the uniparental marker analysis among the Himalayan and adjoining populations reveal the presence of East, Southeast and South Asian genetic signatures. Interestingly, we observed an antagonistic association of Y chromosomal haplogroups O3 and D clines with the longitudinal distance. Thus, we summarise that studying the Himalayan and adjoining populations is essential for a comprehensive reconstruction of the human evolutionary and ethnolinguistic history of eastern Eurasia.

Constitutional mismatch repair deficiency and Lynch syndrome among consecutive Arab Bedouins with colorectal cancer in Israel.

We assessed the molecular characteristics and the frequency of mutations in mismatch-repair genes among Bedouin patients with colorectal cancer (CRC) in Israel. Bedouin patients with a diagnosis of CRC at a major hospital in the southern part of Israel were deemed eligible for this study. The primary screening method was immunohistochemical staining for mismatch-repair proteins (MLH1, MSH2, MSH6, and PMS2). For subjects with abnormal immunohistochemical staining, we performed microsatellite instability (MSI) analyses, and for tumors with a loss of MLH1 expression we also performed BRAF testing. In MSI high cases we searched further for germline mutations. Of the 24 patients enrolled, four subjects (16.7%) had MSI high tumors: one subject was found to harbor a biallelic PMS2 mutation, one subject had Lynch syndrome (LS) with MSH6 mutation and two subjects had a loss of MLH1/PMS2 proteins/BRAF wild type/normal MLH1 sequence. Ten patients (41.7%) were younger than 50 at the time of diagnosis and none had first degree relatives with CRC. In conclusion, in this cohort of 24 consecutive Arab Bedouins with CRC, one patient was found to harbor a constitutional mismatch repair deficiency, one patient had LS with MSH6 mutation, and two patients had unresolved loss of MLH1/PMS2 proteins/BRAF wild type phenotype.

The genetic variation in the R1a clade among the Ashkenazi Levites' Y chromosome.

Approximately 300,000 men around the globe self-identify as Ashkenazi Levites, of whom two thirds were previously shown to descend from a single male. The paucity of whole Y-chromosome sequences precluded conclusive identification of this ancestor's age, geographic origin and migration patterns. Here, we report the variation of 486 Y-chromosomes within the Ashkenazi and non-Ashkenazi Levite R1a clade, other Ashkenazi Jewish paternal lineages, as well as non-Levite Jewish and non-Jewish R1a samples. Cumulatively, the emerging profile is of a Middle Eastern ancestor, self-affiliating as Levite, and carrying the highly resolved R1a-Y2619 lineage, which was likely a minor haplogroup among the Hebrews. A star-like phylogeny, coalescing similarly to other Ashkenazi paternal lineages, ~1,743 ybp, suggests it to be one of the Ashkenazi paternal founders; to have expanded as part of the overall Ashkenazi demographic expansion, without special relation to the Levite affiliation; and to have subsequently spread to non-Ashkenazi Levites.

Nationwide genetic analysis for molecularly unresolved cystic fibrosis patients in a multiethnic society: implications for preconception carrier screening.

BACKGROUND: Preconception carrier screening for cystic fibrosis (CF) is usually performed using ethnically targeted panels of selected mutations. This has been recently challenged by the use of expanded, ethnically indifferent, pan-population panels. Israel is characterized by genetically heterogeneous populations carrying a wide range of CFTR mutations. To assess the potential of expanding the current Israeli preconception screening program, we sought the subset of molecularly unresolved CF patients listed in the Israeli CF data registry comprising ~650 patients. METHODS: An Israeli nationwide genotyping of 152 CF cases, representing 176 patients lacking molecular diagnosis, was conducted. Molecular analysis included Sanger sequencing for all exons and splice sites, multiplex ligation probe amplification (MLPA), and next-generation sequencing of the poly-T/TG tracts. RESULTS: We identified 54 different mutations, of which only 16 overlapped the 22 mutations included in the Israeli preconception screening program. A total of 29/54 (53.7%) mutations were already listed as CF causing by the CFTR2 database, and only 4/54 (7.4%) were novel. Molecular diagnosis was reached in 78/152 (51.3%) cases. Prenatal diagnosis of 24/78 (30.8%) cases could have been achieved by including all CFTR2-causing mutations in the Israeli panel. CONCLUSIONS: Our data reveal an overwhelming hidden abundance of CFTR gene mutations suggesting that expanded preconception carrier screening might achieve higher preconception detection rates.