As the USP domain is widespread across many organisms, there is great diversity in the structures of these proteins. For  Haemophilus influenzae , its UspA resides in the cytoplasm. The protein forms an asymmetric  dimer  with characteristic alpha and beta fold structures. There are differences among different bacteria in areas such as  ATP   binding sites . [ 2 ]  In this case, UspA does not have ATP binding activity. Generally, USPs form dimers and have domains for  nucleotide  binding activity. However, as it is such a diverse group, often with little known about the exact structure, it’s not possible to comment on each USP. In addition to this, UspA may reside in different areas of the cell. For example, in this case it was in the  cytoplasm  but for others, it may be in the  cell membrane . [ 9 ]


Much of the research into USP is done on bacteria, specifically  E. coli  (Strain K-12). Consequently, much is known about the USP domains in bacteria. In  E. coli  there are six families of USP domains which are present in more than 1000 different proteins. [ 10 ]  The six families are Usp A, -C, -D, -E, -F and –G which are triggered by differing environmental insults and often act via varying mechanisms. [ 10 ]


UspA is the most commonly studied USP due to its widespread presence within bacterial genomes. UspA is especially implicated in the resistance of a huge number of stressors most notably tetracycline exposure and high temperatures, with the exception of not forming a response to cold shock. It is thought UspA is especially important to the recovery of  E. coli  following starvation of nutrients. [ 2 ]   UspA during normal growth conditions does not seem to influence gene expression. However, during stressful conditions such as carbon starvation, UspA has been shown to have a global influence on gene expression. A proposed mechanism for such a change in  gene expression  is that UspA has been suggested to bind to DNA. When UspA is mutated,  E. coli  becomes far more vulnerable UV induced DNA damage. [ 11 ]
It’s important to note the USP responses are independent of many other stress responses seen in bacteria such as  rpoS . [ 12 ]


The induction of USP proteins have also been implicated in transitions not only in metabolism or growth but in changes in the colonies' entire phenotype. Bacterial colonies can produce formations known as  biofilms . Zhang and colleagues demonstrated that USPs may be involved in the promotion of intertidal biofilms. [ 13 ]  They observed that during stressful conditions involving metal ions and oxidative stresses that the biofilm phenotype would form. Upon analysis of these biofilms, it could be seen that there was a greatly upregulated level of UspA which Zhang suggests, may be involved with induction of biofilm formation. It is thought UspA may be involved in signalling processes which will upregulate genes involved with biofilm production. [ 12 ]  With findings such as these, it's beginning to be accepted that USPs are acting using an extremely wide range of mechanisms to ensure cell survival.


In bacteria, the USP genes can be regulated by sigma factors within  RNA polymerases . This includes sigma factor σ70 which through binding to a single promoter region, upregulates the transcription of UspA in bacteria. The genes are regulated in a monocistronic fashion. [ 14 ]
Additionally, UspA, UspC, UspD and UspE are over induced during stationary phase through regulation of RecA. RecA is known for its involvement in the repair of DNA via  homologous recombination  following  damage . Consequently, the four Usp domain genes are thought to be mediating the management or protection of DNA. [ 15 ]  Whatever the mechanism exhibited by the proteins, one thing which can be concluded is that USP domains are crucial for survival of many bacterial species. Gomes et al. found that UspA deletions in  Listeria  severely impaired survival as well as listeria’s stress response by  in vitro  and  in vivo . [ 16 ]


USP domain genes are regulated by a number of proteins involved with  growth ,  DNA repair  and  cell division . Notable positive regulation occurs via the action of  ppGpp ,  RecA  and  FtsZ  dependent regulatory pathways. USP domain genes are also under the negative control of FadR.
 [ 17 ]


Plants  contain many hundreds of USP domains and genes. These genes are notably induced by environmental stresses such as  drought . When a lack of  hydration  occurs, biochemical changes induced by the actions of USPs ensue. In response to drought, there is a reduction in  photosynthetic   carbon  production as well as a reduction in  energy metabolism . [ 18 ]  These actions are suggested to occur due to their implications in increasing energy conservation. Water limiting conditions are a common environmental pressure which plants will need to cope with on a regular basis, depending on their habitat. These resistant  phenotypes  will have an increased survival as they allow the plant to conserve energy in times of restricted water which is key to  glucose  production through  photosynthesis . [ 18 ]


Mycobacterium tuberculosis , the infectious agent responsible for  tuberculosis  (TB), persists within an estimated two billion people. TB is known for its ability to transition into a latent state whereby there is slow growth but high persistence within the mammalian host in structures known as  granulomas . [ 19 ]  These granuloma structures are made up of various cellular materials and immune cells. These include macrophages, neutrophils, cellulose and fats. It has long been proposed that USPs play a significant role in the persistence of TB within the human host. This is due to observations of elevated  Usp  genes within  M. tuberculosis  in the latent granuloma stage of the infection. [ 20 ]


There are eight types of USPs within  M. tuberculosis , all of which have an ATP binding domain. It has been found that within  M. tuberculosis , these USPs are regulated by FtsK and FadR. [ 21 ]  One recent finding shows that the induction of USPs within  M. tuberculosis  results in USP binding activity with intracellular cAMP which has indirect implications on transcription within the bacteria. [ 22 ]


Some of  M. tuberculosis'  USPs are suggested to be induced by the  hypoxic  conditions found within the granuloma. Specifically, Rv2623, a type of USP in  M. tuberculosis , is induced by the presence of  nitric oxide ,  reactive oxygen species  and a downshift in  pH . All of these conditions are suggested to be produced by the actions of  macrophages  which are particularly prevalent within the granuloma structures that are characteristic of TB latent infections. [ 20 ]   These conditions have been found to upregulate a particular USP gene called  rv2623 , as well as an additional 50 genes involved in long-term persistence in the mammalian host. It was suggested this USP gene was involved in inducing the latent response within the mammalian host. This stage of the infection is currently chronic with no effective treatments. This makes these kinds of findings extremely valuable.


Rv2623 has an ATP binding domain which if knocked out results in a hyper-virulent form of the bacteria. [ 21 ]  Understanding these processes aids researchers in their quest to provide effective treatment for those suffering from TB. Rv2623 is also a key  biomarker  aiding the diagnostic process for TB. Therefore, these USP genes could be crucial for the long-term survival of the bacteria, meaning that there may be potential therapeutic avenues of research to explore in treating latent TB. [ 23 ]  This comes at a time whereby TB kills many thousands of people a day and is becoming increasing problematic to treat with the rise of  multi-drug-resistant TB .


Similarly, USPs are crucial for the survival of  Salmonella , the causative agent in Salmonellosis. In  developing countries , food poisoning of this kind is a potentially life-threatening condition. The USPs have influence in growth arrest, stress responses and virulence. [ 24 ]  UspA is induced by  metabolic ,  oxidative  and  temperature  related stress. In these conditions UspA is over produced through the  transcriptional regulation  by ppGpp and RecA. These responses have been suggested to be involved in the protection of DNA. As a result, UspA aids Salmonella to resist stressors produced by the mammalian  immune system  assisting in survival and hence, pathogenicity. [ 24 ]  When UspA is inactivated in Salmonella, the mutants die prematurely, demonstrating how crucial these proteins are to survival and persistence. Again, understanding these processes may aid researchers in developing effective drugs to treat these infections. [ 24 ]


An  upstream activating sequence  or  upstream activation sequence  (UAS) is a  cis-acting regulatory sequence  found in  yeast  like  Saccharomyces cerevisiae . It is distinct from the  promoter  and increases the  expression  of a neighbouring  gene . Due to its essential role in activating transcription, the upstream activating sequence is often considered to be analogous to the function of the  enhancer  in multicellular eukaryotes. [ 1 ]  Upstream activation sequences are a crucial part of induction, enhancing the expression of the protein of interest through increased transcriptional activity. [ 2 ]  The upstream activation sequence is found adjacently upstream to a minimal promoter ( TATA box ) and serves as a binding site for  transactivators . If the transcriptional transactivator does not bind to the UAS in the proper orientation then transcription cannot begin. [ 3 ]  To further understand the function of an upstream activation sequence, it is beneficial to see its role in the cascade of events that lead to transcription activation. The pathway begins when activators bind to their target at the UAS recruiting a  mediator . A TATA-binding protein subunit of a  transcription factor  then binds to the TATA box, recruiting additional transcription factors. The mediator then recruits  RNA polymerase II  to the pre-initiation complex. Once initiated, RNA polymerase II is released from the complex and transcription begins. [ 4 ]


The property of the  GAL1-GAL10  to bind the  GAL4  protein is utilised in the  GAL4/UAS technique  for controlled gene mis-expression in Drosophila. This is the most popular form of binary expression in  Drosophila melanogaster , a system which has been adapted for many uses to make  Drosophila melanogaster  one of the most genetically tractable multicellular organisms. [ 5 ]  In this technique, four related binding sites between the  GAL10  and  GAL1  loci in  Saccharomyces cerevisiae  serve as an Upstream Activating Sequences (UAS) element through  GAL4  binding. [ 6 ]  Several studies have been conducted with  Saccharomyces cerevisiae  to explore the exact function of upstream activation sequences, often focusing on the aforementioned  GAL1-GAL10  intergenic region. [ 7 ]  The consensus is 5′-CGG-N 11 -CCG-3′. [ 8 ]


One study explored the galactose-responsive upstream activation sequence (UAS G ), looking at the influence of proximity to this UAS for nucleosome positioning. Proximity to the UAS was chosen because deletions of DNA flanking the UAS left the nucleosome array unaltered, indicating that nucleosome positioning was not related to sequence-specific histone-DNA interactions. The role of specific regions of UAS G  was analyzed by inserting oligonucleotides with different binding properties, leading to the successful identification of a region responsible for the creation of an ordered array. The sequence identified overlapped a binding site for  GAL4  protein, which is a positive regulator for transcription which coincides with the function of upstream activating sequences. [ 9 ]


Another study looked at the effect of inserting the UAS G  into the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD)  [1] . This hybrid promoter was then utilized to express human immune interferon, a toxic substance to yeast that results in a reduced copy number and low plasmid stability. Relative to the native promoter, expression of the hybrid promoter was induced roughly 150- to 200-fold in the cultures by growth in galactose, induction that wasn't apparent with glucose as the carbon source. When compared to the native GPD promoter, the presence of UAS G  caused the transcriptional activity to remain equivalently enhanced under induced conditions. [ 10 ]


The inositol-sensitive upstream activation sequence (UAS INO ) has a  consensus sequence  5'-CATGTGAAAT-3' and is present in the promoter regions of genes that encode enzymes of phospholipid biosynthesis. These enzymes are regulated by inositol and choline, both of which are phospholipid precursors. Within this consensus sequence, the first six bases are homologous with canonical binding motif for proteins within the bHLH or the  basic helix-loop-helix  family. Studies have shown that Ino2p and Ino4p, two bHLH regulatory proteins from  Saccharomyces cerevisiae , bind to promoter fragments containing this element of the consensus sequence. Additional studies have been designed to explore the function of UAS INO  in more detail largely in part because a large number of phospholipid biosynthetic enzyme activities in the model organism  Saccharomyces cerevisiae  show this common pattern of expression. [ 11 ]


One study explored the interaction between Ino4p and Ino2p in more depth, examining the dimerization that takes place between the two prior to binding to the promoter of the  INO 1 gene and activating transcription. By isolating 31 recessive suppressors of the  ino 4-8 mutant of yeast and determining that 29 were of the same locus, the researchers identified the locus as  REG1   [2] . One allele of  REG1 , the suppressor mutant  sia1-1 , was capable of suppressing the inositol auxotrophy, revealing a possible pathway for the repression of inositol-sensitive upstream activating sequence-containing genes of yeast. [ 12 ]


In  microbiology  and  virology , the term  variant  or  genetic variant  is used to describe a subtype of a microorganism that is genetically distinct from a main strain, but not sufficiently different to be termed a distinct  strain . A similar distinction is made in botany between different cultivated varieties of a  species  of plant, termed  cultivars .


It was said in 2013 that "there is no universally accepted definition for the terms 'strain', 'variant', and 'isolate' in the virology community, and most virologists simply copy the usage of terms from others". [ 1 ]  The lack of precise definition continued in 2020; in the context of the  Variant of Concern 202012/01  version of the  SARS-CoV-2  virus, the website of the US  Centers for Disease Control and Prevention  (CDC) states, "For the time being in the context of this variant, the [terms "variant", "strain", and "lineage"] are generally being used interchangeably by the scientific community". [ 2 ]


This  microbiology -related article is a  stub . You can help Wikipedia by  expanding it .


In  genetics ,  virtual karyotype  is the digital information reflecting a  karyotype , resulting from the analysis of short sequences of  DNA  from specific  loci  all over the  genome , which are isolated and enumerated. [ 1 ]  It detects genomic  copy number variations  at a higher resolution for level than conventional karyotyping or chromosome-based  comparative genomic hybridization  (CGH). [ 2 ]  The main methods used for creating virtual karyotypes are  array-comparative genomic hybridization  and  SNP arrays .


A  karyotype  (Fig 1) is the characteristic  chromosome  complement of a  eukaryote   species . [ 3 ] [ 4 ]  A karyotype is typically presented as an image of the chromosomes from a single cell arranged from largest (chromosome 1) to smallest (chromosome 22), with the sex chromosomes (X and Y) shown last. Historically, karyotypes have been obtained by staining cells after they have been chemically arrested during cell division. Karyotypes have been used for several decades to identify chromosomal abnormalities in both germline and cancer cells. Conventional karyotypes can assess the entire genome for changes in chromosome structure and number, but the resolution is relatively coarse, with a detection limit of 5-10Mb. [ citation needed ]


Recently, platforms for generating high-resolution karyotypes  in silico  from disrupted DNA have emerged, such as  array comparative genomic hybridization  (arrayCGH) and  SNP arrays . Conceptually, the arrays are composed of hundreds to millions of probes which are complementary to a region of interest in the genome. The disrupted DNA from the test sample is fragmented, labeled, and hybridized to the array. The hybridization signal intensities for each probe are used by specialized software to generate a log2ratio of test/normal for each probe on the array.  [ citation needed ]


Knowing the address of each probe on the array and the address of each probe in the genome, the software lines up the probes in chromosomal order and reconstructs the genome  in silico  (Fig 2 and 3).


Virtual karyotypes have dramatically higher resolution than conventional cytogenetics. The actual resolution will depend on the density of probes on the array. Currently, the  Affymetrix  SNP6.0 is the highest density commercially available array for virtual karyotyping applications. It contains 1.8 million polymorphic and non-polymorphic markers for a practical resolution of 10-20kb—about the size of a gene. This is approximately 1000-fold greater resolution than karyotypes obtained from conventional cytogenetics. [ citation needed ]


Virtual karyotypes can be performed on germline samples for constitutional disorders, [ 5 ] [ 6 ]  and clinical testing is available from dozens of CLIA certified laboratories ( genetests.org ). Virtual karyotyping can also be done on fresh or formalin-fixed paraffin-embedded tumors. [ 7 ] [ 8 ] [ 9 ]  CLIA-certified laboratories offering testing on tumors include  Creighton Medical Laboratories  (fresh and paraffin embedded tumor samples) and  CombiMatrix Molecular Diagnostics  (fresh tumor samples).


Array-based karyotyping can be done with several different platforms, both laboratory-developed and commercial. The arrays themselves can be genome-wide (probes distributed over the entire genome) or targeted (probes for genomic regions known to be involved in a specific disease) or a combination of both. Further, arrays used for karyotyping may use non-polymorphic probes, polymorphic probes (i.e., SNP-containing), or a combination of both. Non-polymorphic probes can provide only copy number information, while SNP arrays can provide both copy number and loss-of-heterozygosity (LOH) status in one assay. The probe types used for non-polymorphic arrays include cDNA, BAC clones (e.g.,  BlueGnome ), and oligonucleotides (e.g.,  Agilent , Santa Clara, CA, USA or  Nimblegen , Madison, WI, USA). Commercially available oligonucleotide SNP arrays can be solid phase ( Affymetrix , Santa Clara, CA, USA) or bead-based ( Illumina , San Diego, CA, USA). Despite the diversity of platforms, ultimately they all use genomic DNA from disrupted cells to recreate a high resolution karyotype  in silico . The end product does not yet have a consistent name, and has been called virtual karyotyping, [ 8 ] [ 10 ]  digital karyotyping, [ 11 ]  molecular allelokaryotyping, [ 12 ]  and molecular karyotyping. [ 13 ]  Other terms used to describe the arrays used for karyotyping include SOMA (SNP oligonucleotide microarrays) [ 14 ]  and CMA (chromosome microarray). [ 15 ] [ 16 ]  Some consider all platforms to be a type of  array comparative genomic hybridization  (arrayCGH), while others reserve that term for two-dye methods, and still others segregate SNP arrays because they generate more and different information than two-dye arrayCGH methods. [ citation needed ]


Copy number changes can be seen in both germline and tumor samples. Copy number changes can be detected by arrays with non-polymorphic probes, such as arrayCGH, and by SNP-based arrays. Human beings are diploid, so a normal copy number is always two for the non-sex chromosomes. [ citation needed ]


Autozygous  segments and  uniparental disomy  (UPD) are diploid/'copy neutral' genetic findings and therefore are only detectable by SNP-based arrays. Both autozygous segments and UPD will show  loss of heterozygosity  (LOH) with a copy number of two by SNP array karyotyping. The term Runs of Homozgygosity (ROH), is a generic term that can be used for either autozygous segments or UPD. [ citation needed ]


Figure 7 is a SNP array virtual karyotype from a colorectal carcinoma demonstrating deletions, gains, amplifications, and acquired UPD (copy neutral LOH).


A virtual karyotype can be generated from nearly any tumor, but the clinical meaning of the genomic aberrations identified are different for each tumor type. Clinical utility varies and appropriateness is best determined by an oncologist or pathologist in consultation with the laboratory director of the lab performing the virtual karyotype. Below are examples of types of cancers where the clinical implications of specific genomic aberrations are well established. This list is representative, not exhaustive. The web site for the Cytogenetics Laboratory at Wisconsin State Laboratory of Hygiene has additional examples of clinically relevant genetic changes that are readily detectable by virtual karyotyping. [1]


Based on a series of 493  neuroblastoma  samples, it has been reported that overall genomic pattern, as tested by array-based karyotyping, is a predictor of outcome in neuroblastoma: [ 24 ]


Earlier publications categorized neuroblastomas into three major subtypes based on cytogenetic profiles: [ 25 ]


Tumor-specific loss-of-heterozygosity (LOH) for chromosomes 1p and 16q identifies a subset of  Wilms' tumor  patients who have a significantly increased risk of relapse and death. LOH for these chromosomal regions can now be used as an independent prognostic factor together with disease stage to target intensity of treatment to risk of treatment failure. [ 26 ] [ 27 ]


Renal epithelial neoplasms  have characteristic cytogenetic aberrations that can aid in classification. [ 28 ]  See also  Atlas of Genetics and Cytogenetics in Oncology and Haematology .


Array-based karyotyping can be used to identify characteristic chromosomal aberrations in renal tumors with challenging morphology. [ 8 ] [ 10 ]  Array-based karyotyping performs well on paraffin embedded tumors [ 29 ]  and is amenable to routine clinical use.


In addition, recent literature indicates that certain chromosomal aberrations are associated with outcome in specific subtypes of renal epithelial tumors. [ 30 ] Clear cell renal carcinoma: del 9p and del 14q are poor prognostic indicators. [ 31 ] [ 32 ] Papillary renal cell carcinoma: duplication of 1q marks fatal progression. [ 33 ]


Array-based karyotyping is a cost-effective alternative to FISH for detecting chromosomal abnormalities in  chronic lymphocytic leukemia  (CLL). Several clinical validation studies have shown >95% concordance with the standard CLL FISH panel. [ 12 ] [ 34 ] [ 35 ] [ 36 ] [ 37 ]  In addition, many studies using array-based karyotyping have identified 'atypical deletions' missed by the standard FISH probes and acquired uniparental disomy at key loci for prognostic risk in CLL. [ 38 ] [ 39 ]


Four main genetic aberrations are recognized in CLL cells that have a major impact on disease behavior. [ 40 ]


Avet-Loiseau, et al. in  Journal of Clinical Oncology , used SNP array karyotyping of 192  multiple myeloma  (MM) samples to identify genetic lesions associated with prognosis, which were then validated in a separate cohort (n = 273). [ 41 ]  In MM, lack of a proliferative clone makes conventional cytogenetics informative in only ~30% of cases. FISH panels are useful in MM, but standard panels would not detect several key genetic abnormalities reported in this study. [ citation needed ]


Array-based karyotyping cannot detect balanced translocations, such as t(4;14) seen in ~15% of MM. Therefore, FISH for this translocation should also be performed if using SNP arrays to detect genome-wide copy number alterations of prognostic significance in MM. [ citation needed ]


Array-based karyotyping of 260  medulloblastomas  by Pfister S, et al. resulted in the following clinical subgroups based on cytogenetic profiles: [ 42 ]


The 1p/19q co-deletion is considered a "genetic signature" of  oligodendroglioma . Allelic losses on 1p and 19q, either separately or combined, are more common in classic oligodendrogliomas than in either astrocytomas or oligoastrocytomas. [ 43 ]  In one study, classic oligodendrogliomas showed 1p loss in 35 of 42 (83%) cases, 19q loss in 28 of 39 (72%), and these were combined in 27 of 39 (69%) cases; there was no significant difference in 1p/19q loss of heterozygosity status between low-grade and anaplastic oligodendrogliomas. [ 43 ]  1p/19q co-deletion has been correlated with both chemosensitivity and improved prognosis in oligodendrogliomas. [ 44 ] [ 45 ]  Most larger cancer treatment centers routinely check for the deletion of 1p/19q as part of the  pathology  report for oligodendrogliomas. The status of the 1p/19q loci can be detected by FISH or virtual karyotyping. Virtual karyotyping has the advantage of assessing the entire genome in one assay, as well as the 1p/19q loci. This allows assessment of other key loci in glial tumors, such as EGFR and TP53 copy number status. [ citation needed ]


Whereas the prognostic relevance of 1p and 19q deletions is well established for anaplastic oligodendrogliomas and mixed oligoastrocytomas, the prognostic relevance of the deletions for low-grade gliomas is more controversial. In terms of low-grade gliomas, a recent study also suggests that 1p/19q co-deletion may be associated with a (1;19)(q10;p10) translocation which, like the combined 1p/19q deletion, is associated with superior overall survival and progression-free survival in low-grade glioma patients. [ 46 ]  Oligodendrogliomas show only rarely mutations in the p53 gene, which is in contrast to other gliomas. [ 47 ]   Epidermal growth factor receptor  amplification and whole 1p/19q codeletion are mutually exclusive and predictive of completely different outcomes, with EGFR amplification predicting poor prognosis. [ 48 ]


Yin et al. [ 49 ]  studied 55  glioblastoma  and 6 GBM cell lines using SNP array karyotyping. Acquired UPD was identified at 17p in 13/61 cases. A significantly shortened survival time was found in patients with 13q14 (RB) deletion or 17p13.1 (p53) deletion/acquired UPD. Taken together, these results suggest that this technique is a rapid, robust, and inexpensive method to profile genome-wide abnormalities in GBM. Because SNP array karyotyping can be performed on paraffin embedded tumors, it is an attractive option when tumor cells fail to grow in culture for metaphase cytogenetics or when the desire for karyotyping arises after the specimen has been formalin fixed. [ citation needed ]


The importance of detecting acquired UPD (copy neutral LOH) in glioblastoma: [ citation needed ]


In addition, in cases with uncertain grade by morphology, genomic profiling can assist in diagnosis.


Cytogenetics , the study of characteristic large changes in the  chromosomes  of  cancer cells , has been increasingly recognized as an important predictor of outcome in  acute lymphoblastic leukemia  (ALL). [ 52 ] NB: Balanced translocations cannot be detected by array-based karyotyping (see Limitations below).


Some cytogenetic subtypes have a worse prognosis than others. These include:


Correlation of prognosis with bone marrow cytogenetic finding in acute lymphoblastic leukemia


Unclassified ALL is considered to have an intermediate prognosis. [ 56 ]


Myelodysplastic syndrome  (MDS) has remarkable clinical, morphological, and genetic heterogeneity. Cytogenetics play a decisive role in the World Health Organization's classification-based International Prognostic Scoring System (IPSS) for MDS. [ 57 ] [ 58 ]


In a comparison of metaphase cytogenetics, FISH panel, and SNP array karyotyping for MDS, it was found that each technique provided a similar diagnostic yield. No single method detected all defects, and detection rates improved by ~5% when all three methods were used. [ 59 ]


Acquired UPD, which is not detectable by FISH or cytogenetics, has been reported at several key loci in MDS using SNP array karyotyping, including deletion of 7/7q. [ 60 ] [ 61 ]


Philadelphia chromosome–negative  myeloproliferative neoplasms  (MPNs) including polycythemia vera, essential thrombocythemia, and primary myelofibrosis show an inherent tendency for transformation into leukemia (MPN-blast phase), which is accompanied by acquisition of additional genomic lesions.
In a study of 159 cases, [ 62 ]  SNP-array analysis was able to capture practically all cytogenetic abnormalities and to uncover additional lesions with potentially important clinical implications. [ citation needed ]


Identification of biomarkers in  colorectal cancer  is particularly important for patients with stage II disease, where less than 20% have tumor recurrence. 18q LOH is an established biomarker associated with high risk of tumor recurrence in stage II colon cancer. [ 63 ]  Figure 7 shows a SNP array karyotype of a colorectal carcinoma (whole genome view).


Colorectal cancers are classified into specific tumor phenotypes based on molecular profiles [ 63 ]  which can be integrated with the results of other ancillary tests, such as microsatellite instability testing, IHC, and KRAS mutation status:


Malignant rhabdoid tumors  are rare, highly aggressive neoplasms found most commonly in infants and young children. Due to their heterogenous histologic features, diagnosis can often be difficult and misclassifications can occur. In these tumors, the INI1 gene (SMARCB1)on chromosome 22q functions as a classic tumor suppressor gene. Inactivation of INI1 can occur via deletion, mutation, or acquired UPD. [ 64 ]


In a recent study, [ 64 ]  SNP array karyotyping identified deletions or LOH of 22q in 49/51 rhabdoid tumors. Of these, 14 were copy neutral LOH (or acquired UPD), which is detectable by SNP array karyotyping, but not by FISH, cytogenetics, or arrayCGH. MLPA detected a single exon homozygous deletion in one sample that was below the resolution of the SNP array. [ citation needed ]


SNP array karyotyping can be used to distinguish, for example, a medulloblastoma with an isochromosome 17q from a primary rhabdoid tumor with loss of 22q11.2. When indicated, molecular analysis of INI1 using MLPA and direct sequencing may then be employed. Once the tumor-associated changes are found, an analysis of germline DNA from the patient and the parents can be done to rule out an inherited or de novo germline mutation or deletion of INI1, so that appropriate recurrence risk assessments can be made. [ 64 ]


The most important genetic alteration associated with poor prognosis in  uveal melanoma  is loss of an entire copy of  Chromosome 3  ( Monosomy  3), which is strongly correlated with metastatic spread. [ 65 ]  Gains on chromosomes  6  and  8  are often used to refine the predictive value of the Monosomy 3 screen, with gain of 6p indicating a better prognosis and gain of 8q indicating a worse prognosis in  disomy  3 tumors. [ 66 ]  In rare instances, monosomy 3 tumors may duplicate the remaining copy of the chromosome to return to a disomic state referred to as  isodisomy . [ 67 ]  Isodisomy 3 is prognostically equivalent to monosomy 3, and both can be detected by tests for chromosome 3  loss of heterozygosity . [ 68 ]


Unlike karyotypes obtained from conventional cytogenetics, virtual karyotypes are reconstructed by computer programs using signals obtained from  disrupted  DNA. In essence, the computer program will correct translocations when it lines up the signals in chromosomal order. Therefore, virtual karyotypes cannot detect balanced  translocations  and  inversions . They also can only detect genetic aberrations in regions of the genome that are represented by probes on the array. In addition, virtual karyotypes generate a  relative  copy number normalized against a diploid genome, so tetraploid genomes will be condensed into a diploid space unless renormalization is performed. Renormalization requires an ancillary cell-based assay, such as FISH, if one is using arrayCGH. For karyotypes obtained from SNP-based arrays, tetraploidy can often be inferred from the maintenance of heterozygosity within a region of apparent copy number loss. [ 22 ]  Low-level mosaicism or small subclones may not be detected by virtual karyotypes because the presence of normal cells in the sample will dampen the signal from the abnormal clone. The exact point of failure, in terms of the minimal percentage of neoplastic cells, will depend on the particular platform and algorithms used. Many copy number analysis software programs used to generate array-based karyotypes will falter with less than 25–30% tumor/abnormal cells in the sample. However, in oncology applications this limitation can be minimized by tumor enrichment strategies and software optimized for use with oncology samples. The analysis algorithms are evolving rapidly, and some are even designed to thrive on 'normal clone contamination', [ 69 ]  so it is anticipated that this limitation will continue to dissipate.


The  Weismann barrier , proposed by  August Weismann , is the strict distinction between the "immortal" germ cell lineages producing gametes and "disposable" somatic cells in animals (but not plants), in contrast to  Charles Darwin 's proposed  pangenesis  mechanism for inheritance. [ 1 ] [ 2 ]


In more precise terminology, hereditary information is copied only from  germline  cells to  somatic cells . This means that new information from somatic mutation is not passed on to the germline. This  barrier  concept implies that somatic mutations are not inherited. [ 3 ] [ 4 ]


Weismann set out the concept in his 1892 book ″Das Keimplasma: eine Theorie der Vererbung″ (German for  The  Germ Plasm : a theory of inheritance ). [ 5 ]  The use of this theory, commonly in the context of the  germ plasm  theory of the late 19th century, before the development of better-based and more sophisticated concepts of genetics in the early 20th century, is sometimes referred to as  Weismannism . [ 6 ]  Some authors distinguish  Weismannist development  (either  preformistic  or  epigenetic ) that in which there is a distinct germline, from   somatic embryogenesis . [ 7 ]  This type of development is correlated with the  evolution of death  of the somatic line.


The Weismann barrier was of great importance in its day and among other influences it effectively banished certain  Lamarckian  concepts: in particular, it would make Lamarckian inheritance from changes to the body (the soma) difficult or impossible. [ 8 ]  It remains important, but has however required qualification in the light of modern understanding of  horizontal gene transfer  and some other genetic and histological developments. [ 9 ]


The Russian biologist and historian  Zhores A. Medvedev , reviewing Weismann's theory a century later, considered that the accuracy of  genome  replicative and other synthetic systems alone could not explain the "immortal"  germ cell lineages  proposed by Weismann. Rather Medvedev thought that known features of the biochemistry and genetics of  sexual reproduction  indicated the presence of unique information maintenance and restoration processes at the different stages of  gametogenesis . In particular, Medvedev considered that the most important opportunities for information maintenance of  germ cells  are created by  recombination during meiosis  and  DNA repair ; he saw these as processes within the germ cells that were capable of restoring the integrity of  DNA  and  chromosomes  from the types of damage that caused irreversible ageing in  somatic cells . [ 10 ]


Basal animals  such as sponges ( Porifera ) and corals ( Anthozoa ) contain multipotent stem cell lineages, that give rise to both somatic and reproductive cells. The Weismann barrier appears to be of a more recent evolutionary origin among animals. [ 11 ]


In plants, genetic changes in somatic lines can and do result in genetic changes in the germ lines, because the germ cells are produced by somatic cell lineages (vegetative  meristems ), which may be old enough (many years) to have accumulated multiple mutations since seed germination, some of them subject to natural selection. [ 12 ]  It is noteworthy in this context that, generally speaking, adult, reproducing plants tend to produce many more offspring in number than animal organisms.


X-linked recessive inheritance  is a mode of  inheritance  in which a  mutation  in a  gene  on the  X chromosome  causes the  phenotype  to be always expressed in males (who are necessarily  hemizygous  for the gene mutation because they have one X and one  Y chromosome ) and in females who are homozygous for the gene mutation, see  zygosity . Females with one copy of the mutated gene are carriers.


X-linked inheritance means that the gene causing the trait or the disorder is located on the X chromosome. Females have two X chromosomes while males have one X and one  Y chromosome . Carrier females who have only one copy of the mutation do not usually express the phenotype, although differences in  X-chromosome inactivation  (known as  skewed X-inactivation ) can lead to varying degrees of clinical expression in carrier females, since some cells will express one X  allele  and some will express the other. The current estimate of sequenced X-linked genes is 499, and the total, including vaguely defined traits, is 983. [ 1 ]


In humans, inheritance of X-linked recessive traits follows a unique pattern made up of three points.


A few scholars have suggested discontinuing the use of the terms  dominant  and  recessive  when referring to  X-linked inheritance . [ 5 ]  The possession of two X chromosomes in females leads to dosage issues which are alleviated by  X-inactivation . [ 6 ]  Stating that the highly variable  penetrance  of X-linked traits in females as a result of mechanisms such as skewed X-inactivation or  somatic mosaicism  is difficult to reconcile with standard definitions of dominance and recessiveness, scholars have suggested referring to traits on the X chromosome simply as  X-linked. [ 5 ]


The most common X-linked recessive disorders are: [ 7 ]


Theoretically, a mutation in any of the  genes on chromosome X  may cause disease, but below are some notable ones, with short description of symptoms:


[Female X-linked disorders]


Ribosomopathies  are diseases caused by abnormalities in the structure or function of  ribosomal  component  proteins  or  rRNA   genes , or other genes whose products are involved in  ribosome biogenesis . [ 1 ] [ 2 ] [ 3 ]


Ribosomes  are essential for  protein synthesis  in all living organisms.  Prokaryotic  and  eukaryotic  ribosomes both contain a  scaffold  of  ribosomal RNA  (rRNA) on which are arrayed an extensive variety of  ribosomal proteins  (RP). [ 4 ]  Ribosomopathies can arise from abnormalities of either rRNA or the various RPs. [ citation needed ]


The nomenclature of rRNA subunits is derived from each component's  Svedberg unit , which is an  ultracentrifuge   sedimentation coefficient , that is affected by mass and also shape. These S units of the rRNA subunits cannot simply be added because they represent measures of sedimentation rate rather than of mass.  Eukaryotic ribosomes  are somewhat larger and more complex than prokaryotic ribosomes. The overall 80S eukaryotic  rRNA structure is composed of a  large 60S subunit  (LSU) and a  small 40S subunit  (SSU). [ 5 ]


In humans, a single transcription unit separated by 2  internally transcribed spacers  encodes a precursor,  45S . The precursor 45S rDNA is organized into 5 clusters (each has 30-40 repeats) on chromosomes 13, 14, 15, 21, and 22. These are transcribed in the  nucleolus  by  RNA polymerase I . 45S is processed in the nucleus via 32S rRNA to  28S [ 6 ]  and  5.8S , [ 7 ]  and via 30S  to  18S , [ 8 ]  as shown in the diagram. 18S is a component of the ribosomal 40S subunit. 28S, 5.8S and  5S , [ 9 ]  which is transcribed independently, are components of 60S. The 5S DNA occurs in  tandem arrays  (~200-300 true 5S genes and many dispersed  pseudogenes ); the largest is on chromosome 1q41-42. 5S rRNA is transcribed by  RNA polymerase III . [ 5 ]  It is not fully clear why rRNA is processed in this way rather than being directly transcribed as mature rRNA, but the sequential steps may have a role in the proper folding of rRNA or in subsequent RP assembly.


The products of this processing within the  cell nucleus  are the four principal types of cytoplasmic rRNA: 28S, 5.8S, 18S, and 5S subunits. [ 10 ] : 291   and (cite)(cite) (Mammalian cells also have 2 types of mitochondrial rRNA molecules,  12S  and  16S .) In humans, as in most eukaryotes, the  18S  rRNA is a component of 40S ribosomal subunit, and the 60S large subunit contains three rRNA species (the  5S ,  5.8S  and  28S  in mammals, 25S in plants). 60S rRNA acts as a  ribozyme , catalyzing  peptide bond formation , while 40S monitors the  complementarity  between  tRNA   anticodon  and  mRNA . [ citation needed ]


Abnormal  ribosome biogenesis  is linked to several human  genetic diseases . [ citation needed ]


Ribosomopathy has been linked to skeletal muscle atrophy, [ 11 ]  and underpins most  Diamond–Blackfan anemia  (DBA), [ 2 ]  the  X-linked  subtype of  dyskeratosis congenita  (DKCX), [ 12 ] [ 13 ]   Treacher Collins syndrome   (TCS), [ 2 ] [ 14 ]   Shwachman–Diamond syndrome  (SDS) [ 15 ]  and  5q-   myelodysplastic syndrome .(5q- MDS),(cite)(cite)  North American Indian childhood cirrhosis  (NAIC), [ 16 ]   isolated congenital asplenia  (ICAS), [ 16 ] [ 17 ] [ 18 ] [ 19 ]  and  Bowen–Conradi syndrome  (BWCNS),  CHARGE syndrome [ 20 ] [ 21 ] [ 22 ] [ 23 ]  and  ANE syndrome  (ANES). [ 24 ]


The associated  chromosome ,  OMIM   genotype ,  phenotype , and possible disruption points are shown:


Several ribosomopathies share features such as  inherited bone marrow failure , which is characterized a reduced number of blood cells and by a predisposition to  cancer . [ 5 ]  Other features can include skeletal abnormalities and growth retardation. [ 16 ]  However, clinically these diseases are distinct, and do not show a consistent set of features. [ 16 ]


With the exception of rare  GATA1  genotypes,(cite)  Diamond–Blackfan anemia  (DBA) arises from a variety of mutations that cause ribosomopathies. [ 35 ]


The  X-linked  subtype of  dyskeratosis congenita  (DKCX) [ citation needed ]


Shwachman–Diamond syndrome  (SDS) is caused by bi-allelic mutations in the  SBDS  protein that affects its ability to couple GTP hydrolysis by the GTPase EFL1 to the release of  eIF6  from the 60S subunit. [ 36 ]  Clinically, SDS affects multiple systems, causing bony abnormalities, and pancreatic and neurocognitive dysfunction. [ 37 ]  SBDS associates with the 60S subunit in human cells and has a role in subunit joining and translational activation in yeast models. [ citation needed ]


5q-   myelodysplastic syndrome  (MDS) [ 37 ]  is associated with acquired haplo-insufficiency of  RPS14 , [ 37 ]  a component of the  eukaryotic small ribosomal subunit (40S) . [ 5 ]  RPS14 is critical for 40S assembly, and depletion of RPS14 in human  CD34 (+) cells is sufficient to recapitulate the 5q- defect of  erythropoiesis  with sparing of  megakaryocytes . [ 5 ]


Treacher Collins syndrome  (TCS)


Cartilage–hair hypoplasia  (CHH) - some sources list confidently as ribosomopathy, others question [ citation needed ]


NAIC is an autosomal recessive abnormality of the  UTP4  gene, which codes for cirhin.  Neonatal jaundice  advances over time to biliary  cirrhosis  with severe liver  fibrosis .


Bowen–Conradi syndrome (BCS [ 38 ]  or BWCNS [ 39 ] ) is an  autosomal recessive  abnormality of the  EMG1   gene , which plays a role in  small ribosomal subunit  (SSU) assembly. [ 38 ] [ 40 ] [ 41 ]  Most affected children have been from North American  Hutterite  families, but BWCNS can affect other population groups. [ 39 ] [ 42 ]  Skeletal dysmorphology is seen [ 39 ] [ 42 ]  and severe prenatal and postnatal growth failure usually leads to death by one year of age. [ 43 ]


Unlike the mutations of the 5 genes associated with  DNA mismatch repair , which are associated with  Lynch syndrome  with  hereditary nonpolyposis colorectal cancer (HNPCC) due to  microsatellite instability , familial colorectal cancer (CRC) type X (FCCX) gives rise to HNPCC despite microsatellite stability. [ 44 ]  FCCX is most likely etiologically heterogeneous but  RPS20  may be implicated in some cases. [ 44 ]


The  p53  pathway is central to the ribosomopathy phenotype. [ 45 ]  Ribosomal stress triggers activation of the p53 signaling pathway. [ 46 ] [ 47 ]


Cancer  cells have irregularly shaped, large  nucleoli , which may correspond ribosomal gene transcription up-regulation, and hence high  cell proliferation .   Oncogenes , like  c-Myc , can upregulate  rDNA  transcription in a direct and indirect fashion. Tumor suppressors like Rb and  p53 , on the other hand, can suppress ribosome biogenesis. Additionally, the nucleolus is an important cellular sensor for stress and plays a key role in the activation of p53.


Ribosomopathy has been linked to the pathology of various malignancies. [ 45 ]  Several ribosomopathies are associated with an increased rate of cancer. For example, both SDS and 5q- syndrome lead to impaired  hematopoiesis  and a predisposition to  leukemia . [ 37 ]  Additionally, acquired defects in ribosomal proteins that have not been implicated in congenital ribosomopathies have been found in  T-lymphoblastic leukemia/lymphoma ,  stomach cancer  and  ovarian cancer . [ 3 ]


Cartilage–hair hypoplasia  ( CHH ) is a rare  genetic disorder . Symptoms may include short-limbed  dwarfism  due to  skeletal dysplasia , variable level of  immunodeficiency , and predisposition to  cancer . It was first reported by  Victor McKusick  in 1965.


CHH is an  autosomal recessive [ 2 ]  inherited disorder. It is a highly  pleiotropic  disorder. A rarely encountered genetic phenomenon, known as  uniparental disomy  (a genetic circumstance where a child inherits two copies of a  chromosome  from one parent, as opposed to one copy from each parent) has also been observed with the disorder. [ 2 ]


An association between mutations near or within the  ncRNA  component of RNase MRP,  RMRP , has been identified. [ 3 ] [ 4 ] [ 5 ] [ 6 ]  The endoribonuclease RNase MRP is a complex of RNA molecule and several proteins and it participates in cleavage of mitochondrial primers responsible for DNA replication and in pre-rRNA processing in the nucleolus. [ 7 ] [ 8 ]  The locus of the gene has been mapped to the short arm of chromosome 9. [ 9 ]


Patients with CHH usually suffer from cellular immunodeficiency. In the study of 108 Finnish patients with CHH, there was detected mild to moderate form of  lymphopenia , decreased delayed type of hypersensitivity and impaired responses to  phytohemagglutinin . [ 10 ]  This leads to susceptibility to and, in some more severe cases, mortality from infections early in childhood. There has also been detected combined immunodeficiency in some patients. [ 11 ]
Patients with CHH often have increased predispositions to malignancies. [ 11 ]


A verified treatment for this disease is yet to be discovered. [ citation needed ]


Diamond–Blackfan anemia  ( DBA ) is a  congenital   erythroid   aplasia  that usually presents in  infancy . [ 3 ]  DBA causes low  red blood cell  counts ( anemia ), without substantially affecting the other blood components (the  platelets  and the  white blood cells ), which are usually normal. This is in contrast to  Shwachman–Bodian–Diamond syndrome , in which the  bone marrow  defect results primarily in  neutropenia , and  Fanconi anemia , where all cell lines are affected resulting in  pancytopenia . There is a risk to develop acute myelogenous leukemia (AML) and certain other cancers. [ 4 ]


A variety of other congenital abnormalities may also occur in DBA, such as hand anomalies.


Diamond–Blackfan anemia is characterized by  normocytic  or  macrocytic   anemia  (low  red blood cell  counts) with decreased erythroid  progenitor cells  in the  bone marrow .  This usually develops during the  neonatal  period.  About 47% of affected individuals also have a variety of  congenital  abnormalities, including  craniofacial  malformations, thumb or upper limb abnormalities, cardiac defects,  urogenital  malformations, and  cleft palate . [ 5 ]   Low birth weight and generalized growth delay are sometimes observed. DBA patients have a modest risk of developing  leukemia  and other malignancies. [ 6 ]


Most  pedigrees  suggest an  autosomal dominant  mode of inheritance [ 1 ]  with incomplete  penetrance . [ 7 ]   Approximately 10–25% of DBA occurs with a family history of disease.


~70% of DBA cases can be attributed genetic mutations affecting  ribosomal  protein  genes . [ 8 ]  The disease is characterized by genetic  heterogeneity , affecting different ribosomal gene loci: [ 9 ]  Exceptions to this paradigm have been demonstrated, such as with rare mutations of transcription factor  GATA1 . [ 10 ] [ 11 ]   RPS19 ,  RPL5 ,  RPS26 , and  RPL11  are the most frequently mutated genes in DBA patients. [ 8 ]  Given that ribosome function is essential for life, DBA patients carry loss-of-function alleles affecting only one copy. Initial descriptions of DBA patients primarily concentrated on nonsense and missense mutations within ribosomal protein coding sequences. However, recent findings suggest that extended splice site variations have not been sufficiently recognized and are quite common. [ 8 ]  Recent studies have begun to characterize the molecular signatures associated with specific mutations that lead to aberrant splicing impacting ribosomal proteins such as RPL11. [ 12 ]


SLC49A1
( FLVCR1 )


In 1997, a patient was identified who carried a rare balanced  chromosomal translocation  involving  chromosome 19  and the  X chromosome .  This suggested that the affected gene might lie in one of the two regions that were disrupted by this  cytogenetic   anomaly .   Linkage  analysis in affected families also implicated this region in disease, and led to the cloning of the first DBA gene.  About 20–25% of DBA cases are caused by mutations in the  ribosome  protein S19 ( RPS19 ) gene on chromosome 19 at  cytogenetic  position 19q13.2.  Some previously undiagnosed relatives of DBA patients were found to carry mutations, and also had increased adenosine deaminase levels in their red blood cells, but had no other overt signs of disease. [ citation needed ]


A subsequent study of families with no evidence of  RPS19  mutations determined that 18 of 38 families showed evidence for involvement of an unknown gene on chromosome 8 at 8p23.3-8p22. [ 22 ]   The precise genetic defect in these families has not yet been delineated.


Malformations are seen more frequently with DBA6  RPL5  and DBA7  RPL11  mutations. [ 7 ]


The genetic abnormalities underpinning the combination of DBA with  Treacher Collins syndrome  (TCS)/ mandibulofacial dysostosis  (MFD) phenotypes are heterogeneous, including  RPS26  (the known DBA10 gene),  TSR2  which encodes a direct binding partner of  RPS26 , and  RPS28 . [ 21 ]


The phenotype of DBA patients suggests a  hematological   stem cell  defect specifically affecting the erythroid progenitor population.  Loss of ribosomal function might be predicted to affect  translation  and  protein biosynthesis  broadly and impact many tissues.  However, DBA is characterized by dominant inheritance, and arises from partial loss of ribosomal function, so it is possible that erythroid progenitors are more sensitive to this decreased function, while most other tissues are less affected. [ citation needed ]


Typically, a diagnosis of DBA is made through a  blood count  and a  bone marrow biopsy .


A diagnosis of DBA is made on the basis of anemia, low  reticulocyte  (immature red blood cells) counts, and diminished erythroid precursors in bone marrow.  Features that support a diagnosis of DBA include the presence of congenital abnormalities,  macrocytosis , elevated  fetal hemoglobin , and elevated  adenosine deaminase  levels in red blood cells. [ 23 ]


Most patients are diagnosed in the first two years of life.  However, some mildly affected individuals only receive attention after a more severely affected family member is identified. [ citation needed ] About 20–25% of DBA patients may be identified with a  genetic test  for mutations in the  RPS19  gene.


Corticosteroids  can be used to treat anemia in DBA.  In a large study of 225 patients, 82% initially responded to this therapy, although many side effects were noted. [ 24 ]   Some patients remained responsive to steroids, while  efficacy  waned in others.   Blood transfusions  can also be used to treat severe anemia in DBA.  Periods of  remission  may occur, during which transfusions and steroid treatments are not required.   Bone marrow transplantation  (BMT) can cure hematological aspects of DBA.  This option may be considered when patients become transfusion-dependent because frequent transfusions can lead to iron overloading and organ damage.  However,  adverse events  from BMTs may exceed those from iron overloading. [ 25 ]  A 2007 one-subject study [ 26 ]  showed the potential efficacy of leucine and isoleucine supplementation.


First noted by Hugh W. Josephs in 1936, [ 1 ] [ 27 ]  the condition is however named for the pediatricians  Louis K. Diamond  and  Kenneth Blackfan , who described congenital  hypoplastic  anemia in 1938. [ 28 ]   Responsiveness to  corticosteroids  was reported in 1951. [ 1 ]  In 1961, Diamond and colleagues presented  longitudinal  data on 30 patients and noted an association with skeletal abnormalities. [ 29 ]  In 1997, a region on chromosome 19 was determined to carry a gene mutated in some DBA. [ 30 ] [ 31 ]  In 1999, mutations in the  ribosomal protein S19  gene ( RPS19 ) were found to be associated with disease in 42 of 172 DBA patients. [ 32 ]   In 2001, a second DBA gene was localized to a region of chromosome 8, and further genetic heterogeneity was inferred. [ 33 ]  Additional genes were subsequently identified. [ 9 ]


North American Indian childhood cirrhosis  ( NAIC ) is a disease in humans that can affect  Ojibway-Cree  children in northwestern  Quebec , Canada. [ 1 ]  The disease is due to an  autosomal recessive  abnormality of the  UTP4   gene , which codes for cirhin, [ 2 ]  a  nucleolar  protein. [ 3 ]


NAIC is a  ribosomopathy . [ 4 ] [ 5 ]  An R565W  mutation  of  UTP4 [ 2 ] [ 6 ]  leads to partial impairment of cirhin interaction with  NOL11 . [ 6 ]


Initial transient neonatal jaundice advances over time to  biliary cirrhosis  with severe liver  fibrosis . [ 1 ]  Eventually,  liver failure  occurs, and requires liver  transplantation . [ 1 ]


The  Bulgarians  are part of the  Slavic  ethnolinguistic group as a result of migrations of Slavic tribes to the region since the 6th century AD and the subsequent linguistic assimilation of other populations. [ 2 ] [ 3 ] [ 4 ] [ 5 ]


Hellenthal et al., 2014  estimated from data of 94 modern populations a couple of analyses on Bulgarians inferred from an admixture event in 1000-1600 YBP between a  Slavic  and a  Cypriot  donor group: to a Polish donor group Bulgarians are of an estimated 59%  Polish -like and 41%  Cypriot -like admixture; to a Belarusian, Bulgarians are of 46%  Belarusian -like and 54%  Cypriot -like admixture. [ 6 ] [ 7 ]  Early gene flows between southeastern and eastern Europe make it difficult to obtain a correct estimate, although young enough identical by descent segments confirmed such connection and that the East and West Slavs share more identical by descent segments with South Slavs than with  Greeks , inter-Slavic populations (a group of  Romanians ,  Gagauz ), but less with  Balts , while the South Slavs share similar number with East and West Slavs, but fewer with Greeks. [ 8 ]


The phenomenon of distinct genetic substrata in the West, East, and South Slavs would imply several mechanisms, including cultural assimilation of indigenous populations by bearers of Slavic languages as a major mechanism of the spread of Slavic languages to the Balkan Peninsula. [ 8 ] [ 9 ]  About 55% of the Bulgarian autosomal genetic legacy is Mediterranean, about the half of which resembles the Caucasian, Middle Eastern and to a lesser extent the North African genetics. [ 7 ]  Combining all lines of evidence, it is suggested that the major part of the within-Balto-Slavic genetic variation can be mainly attributed to the assimilation of the pre-existing regional genetic components, which differ for West, East and South Slavic-speaking peoples. [ 8 ]


Around 4% of Bulgarian genes are derived outside of Europe and the Middle East or are of undetermined origin (by 858 CE), of which 2.3% are from  Northeast Asia  and correspond to Asian tribes such as  Bulgars , [ 10 ]  a consistent very low frequency for  Eastern Europe  as far as  Uralic-speaking   Hungarians .


The national groups of Eastern Europe are characterized by dominant haplogroups  R1a  or  I2 , while those of Western Europe are characterized by dominant haplogroups  R1b  or  I1 , and by other dominant haplogroups in Europe are characterized Albanians, Finns, Greeks, and Turks, and over 50% of the total pool of each European nation other than the latter consists of these 4 haplogroups. [ 8 ]  A consistent pattern of both all national and regional groups of Bulgarians and  Balto-Slavs  is that their sum of haplogroups R1a and I2 is always larger than their sum of R1b and I1, while this ratio is vice versa in the Europeans not native to the Post- Eastern Bloc . [ 8 ]  Bulgarians, as some of their neighbours show the highest diversity of haplogroups in Europe, marked by significant (> 10%) frequencies of 5 major haplogroups. Most Bulgarians belong to three unrelated haplogroups, 20% of whom to  I-M423  (I2a1b), 18% to  E-V13  (E1b1b1a1b1a) and 17% to  R-Z282  (R1a1ab1), but the biggest part belongs to macro-haplgoroup R (~28%). The major haplogroups, grouped by age of around 20 kya, are: [ 11 ]


Haplogroup frequencies of Bulgarians


A phylogenetic analysis determines that the population of Haskovo Province has shorter genetic distance against the population of the Czech Republic than to the Bulgarian provinces, and that only the population of Burgas Province is closer to Haskovo than the Hungarian population, furthermore only datasets of two more Balkan or Slavic foreign populations(Greece and Croatia) are used and all other Slavic populations are excluded from this analysis. [ 46 ]  The largest-scale study of the Hungarians (n=230) determined that [ 47 ]  the remaining Finno-Urgic peoples are genetically their furthest populations, and clearly confirmed that the closest Europeans to the Hungarians are the Bulgarians, however the same study determines the  Yugoslavs  as the nearest population to Bulgarians. [ 48 ]  According to DNA data for 17 Y-chromosomal  STR loci  in Macedonians, the Macedonian population has the lowest genetic distance against the Bulgarian population (0.0815). [ 49 ]


According to an older study of 127 Bulgarian males, frequencies are the following: 30% R (17% R1a, 11% R1b, 2% R*); 27.5% I; 20% E; 18% J; 1.5% G; 1.5% H; 1% T. [ 50 ]


According to another study involving 126 Bulgarian males, frequencies are the following: 30% I (25.5% I2a, 4% I1); 20,5% E; 17.5% R (R1b 11%, R1a 6%); 17.5% J (16% J2); 5.5% G; 4% Q; 1% L; 1% T; unknown 3%. [ 51 ]


According to another study involving 100 Bulgarian males, frequencies are the following: 34% I (29% I2a, 3% I1); 30% R (16% R1a, 14% R1b); 21% E (20% E1b1b1a); 9% J; 2% G; 2% T; 1% N. [ 52 ]


Complementary evidence exists from  mtDNA  data. Bulgaria shows a very similar profile to other European countries – dominated by mitochondrial haplogroups  Hg H  (~42%),  Hg U  (~18%),  Hg J / Hg T  (~18%), and  Hg K  (~6%). [ 53 ]  Like most Europeans, H1 is the prevailing subclade among Bulgarians. [ 54 ]  Most of the U-carriers belong to U5 and U4. The distribution of the subclades of Haplogroup H have not been revealed. Recent studies show greater diversity within mt Haplogroups than once thought, as sub-haplogroups are being discovered, and often separate migrations and distributions of the Y-DNA haplogroups. While the Y-DNA variation in Europe is clinal, the mitochondrial is not. [ 55 ]


MtDNA haplgroups of ~1000 Bulgarians: [ 53 ]


Whilst haploid markers such as mtDNA and Y-DNA can provide clues about past population history, they only represent a single genetic locus, compared to hundreds of thousands present in nuclear, autosomes. Although autosomal analyses often sample a small number of Bulgarians, by multiple autosomes multiple ancestral lines may be traced by an individual's 21 autosomes as opposed to one identical mtDNA or Y-DNA sex chromosome, whose inheritance although clinal, demonstrates  genetic drift  often in statistics. Analyses of autosomal DNA markers gives the best approximation of overall 'relatedness' between populations, presenting a less skewed genetic picture compared to Y-DNA haplogroups. This auDNA data shows that there are no sharp discontinuities or clusters within the European population. Rather there exists a genetic gradient, running mostly in a southeast to northwest direction. A study compared all Slavic nations and combined all lines of evidence,  autosomal , maternal and paternal, including more than 6000 people for and at least 700 Bulgarians from previous studies, of which 13 were used for autosomal analysis (right image). The overall data situates the southeastern group (Bulgarians and Macedonians) in a cluster with  Romanians , and they are at similar proximity to  Gagauzes ,  Montenegrins  and  Serbs  who are not part of another cluster but are described as 'in between' clusters. [ 8 ]  Macedonians and Romanians consistently appear to be among the most related to Bulgarians by au, mt, and Y-DNA [ 8 ]  a conclusion backed also by a pan-European autosomal study investigating 500,568 SNP (loci) of 1,387 Europeans and including 1 or 2 Bulgarians, [ 56 ]  other more or less extensive data sets situate Bulgarians and Romanians as their nearest . [ 57 ] [ 58 ] [ 59 ]  Per  HLA-DRB1  allele frequencies Bulgarians are also in a cluster with the same populations. [ 60 ]  The Balto-Slavic study itself calculated genetic distance by SNP data of the multiple autosomes and ranked most proximal to Bulgarians the Serbs, followed by Macedonians, Montenegrins, Romanians, Gagauzes,  Macedonian Greeks  apart from  Thessaloniki , the rest of the  South Slavs , Hungarians, Slovaks, Czechs, and then by Greeks from  Thessaloniki ,  Central Greece  and  Peloponnese . [ 8 ]  The  East Slavs  and Poles cluster together remaining less proximal to Bulgarians than  Germans , among whom Slav admixture is also observed.  Balts , however, according to the  PCA analysis  are less proximal to Bulgarians than Italians for example are. [ 8 ]  Bulgarians are also only modestly close to their eastern neighbours – the Anatolian Turks, suggesting the presence of certain geographic and cultural barriers between them. [ 61 ]  Despite various invasions of Altaic-speaking peoples in Europe, no significant impact from such Asian descent is recorded throughout southern and central Europe. [ 62 ]


The study claims that the major part of the  Balto-Slavic  genetic variation can be primarily attributed to the assimilation of the pre-existing regional genetic components, which differed for present West, East and South Slavic-speaking people. [ 8 ]  For Slavic peoples correlations with linguistics came out much lower than high correlations with geography. [ 8 ]  The  South Slavic  group, despite sharing a common language, is separated and has a largely different genetic past from their  northern linguistic relatives . [ 5 ] [ 8 ] [ 9 ]  Therefore, for the Bulgarians and most other  South Slavs  the most plausible explanation would be that their most sizable genetic components were inherited from indigenous  Balkan  pre-Slavic and pre-Bulgar population. [ 5 ] [ 8 ] [ 9 ]  The South Slavs are characterized by featuring NRY hgs  I2a  and  E  plus 10% higher Mediterranean k2 autosomal component, while the Eastern and Western Slavs are characterized by the k3 component and hg  R1a . [ 8 ]  The presence of two distinct genetic substrata in the genes of East-West and South Slavs would conclude that assimilation of indigenous populations by bearers of Slavic languages was a major mechanism of the spread of Slavic languages to the Balkan Peninsula. [ 8 ]


Southeastern Europeans share large numbers of common ancestors that date roughly to the times of the Slavic expansion around 1,500 years ago. The eastern European populations with high rates of ( IBD ) are highly coincident with the modern distribution of Slavic languages including Hungary, Romania, Greece and Albania, so it is speculated for Slavic expansion, anyway it was concluded that additional work and methods would be needed to verify this hypothesis. This study detects a considerable connection between Bulgarians and North Slavs that is the result of migrations no earlier than 1500 years ago. [ 59 ]  A study on genetic admixture filtered to 474,491 autosomal SNPs and including 18 Bulgarians concluded that there is a recent excess of identical by descent sharing in Eastern Europe, and recent period of exchanged segments speculating that this may correspond to the Slavic expansion across this region. A signal at a low frequency among Balkan Slavs was detected that may have been inherited from the medieval Slavic settlers, but it was confirmed that this issue requires further investigation. [ 8 ]  The short genetic distance of South Slavs does not extend to populations throughout the whole Balkan Peninsula and they are differentiated from all Greek sub-populations that are not  Macedonian Greek . [ 8 ]  The South Slavs share significantly fewer identical by descent segments for length classes with Greeks than with the group of East-West Slavs. [ 8 ]  Most of the East-West Slavs share as many such segments with the South Slavs as they share with the inter-Slavic populations between them. This might suggest Slavic gene flow across the wide area and physical boundaries such as the Carpathian Mountains, including Hungarians, Romanians and Gagauz. [ 8 ]  Notably, the number of common ancestors within the last 1,000 to 2,000 years is particularly high within eastern and Slavic-speaking Europe. [ 63 ]  A high number of shared IBD segments among East Europeans that can be dated to around 1,000–2,000 YBP was revealed. The highest percentage of the total number of shared pairwise IBD segments is detected between the group of East-West Slavs and South Slavs (41% from the total number of IBD segments detected); Baltic speakers, Estonians (40%) and "inter-Slavic" Hungarians, Romanians and Gagauz (37%). East-West Slavs share these segments with Western Europeans (32%), Volga region populations (30) and North Caucasus (21%). South Slavs also share 41% with East-West Slavs and 37% with Inter-Slavic populations, they also share 31% with Western Europeans and 30% with Greeks. However, per one pair of individuals East-West Slavs share more IBD with Balts than with South Slavs, but not with the rest and the same amount with inter-Slavic as with South Slavs. Per one pair of individuals South Slavs keep sharing most IBD with East-South Slavs and the same amount with the inter-Slavic, followed by Greeks and Western Europeans. [ 8 ]


For Bulgarians the prevailing donor group in admixture with up to more than 40% are a northeastern group, consistent with the medieval Slav expansion, the date of the admixture event is set at 900-1300 CE. The Slavic frequency of the Bulgarians is determined lower than that of Poles and Hungarians, higher than that of Greeks and roughly the same as Romanians. [ 7 ]  In the publication by  Hellenthal et al. , based on a database of 94 modern populations, the authors inferred over 40% of the total  autosomal  make-up of the Bulgarians to a legacy of the "Slavic (500-900CE)" expansion. [ 7 ]  The same publication provided the following two analyses on oneway admixture of the Bulgarian  autosomal  makeup:
1) 46.0%  Belarusian -like (including 23.2%  Lithuanian , 19.3%  Polish , 2.3%  Finnish ) and 54.0%  Cypriot -like (including 14.8%  Greek , 12.7%  Cypriot , 11.9%  Arab , 4.1%  Italian , 3.3%  Georgian , 2.0%  Sardinian , 2.8%  Iranian )
2) 59.0%  Polish -like (including 54.5%  Polish , 4.0%  English ) and 40.9%  Cypriot -like (including 15.3%  Cypriot , 14.9%  Arab , 3.5%  Georgian , 2.4%  Sardinian , 1.4%  Hezhen , 1.3%  Greek ). [ 6 ]


The genetic diversity among Bulgarians is the reason of more inherited diseases [ 64 ]  The  blood type  of 21,568 Bulgarians is 37% A+, 28% 0+, 14% B+, 7% AB+, 6+ A−, 4% 0−, 2% B−, 1% AB−, [ 65 ]  a  distribution  similar to Sweden, the Czech Republic and Turkey.


Despite the most common haplogroup among Bulgarians being 14000 years old [ 66 ]  I2a1b at 20%, 8000 years old hunter-gatherer samples of the same haplogroup came out genetically very distant from Bulgarian and Balkan individuals by an  autosomal  analysis of skeletal remains from Loschbour cave in Luxembourg. [ 67 ]


Computing the frequency of common point mutations of several mtDNA Thracian remains from South-East of Romania with haplogroups H17, H22 and HV has resulted that the Italian (7.9%), the Albanian (6.3%) and the Greek (5.8%) have shown a bias of closer genetic kinship with the Thracian individuals than the Romanian and Bulgarian individuals (4.2%), but it was noted that more mtDNA sequences from Thracian individuals are needed in order to perform a complex objective statistical analysis. [ 68 ]  From seven Thracian samples aged about 3 millennia from Gabova Mogila and Shekerdja Mogila in  Sliven Province , and from Bereketska Mogila in  Stara Zagora Province , two were identified as belonging to mtDNA  Haplogroup D , presumably associated with East Asia. Haplogroup W5a was found among two individuals and H1an2. H14b1 was also found. [ 69 ]  Four samples from Iron Age Bulgaria were studied, the official study confirmed only that the two are male and mtDNA of two individuals - U3b for the  Svilengrad  man and HV for the  Stambolovo  individual. Haplogroups U for the  Krushare  man, U2e for the  Vratitsa  individual have been identified. Those individuals were from Thracian burial sites and are dated at around 450-1500 BC. [ 70 ]


20 samples from medieval Bulgarian sites were alleged as originally Bulgar, but there is no evidence for that. [ 71 ]  They were from a burial site from the Monastery of  Mostich  in  Preslav ,  Nozharevo ,  Tuhovishte  and most came out European mtDNA haplogroup H, including H1, H1an2, H1r1, H1t1a1, H2a2a1 H5, H13a2c1, H14b1, HV1, J, J1b1a1, T, T2, U4a2b, U4c1 and U3 with the half belonging to Haplogroup H. It was shown a short genetic distance between these samples and modern Bulgarians. [ 72 ]


After at least 20 mediaval(10-14th century) mtDNA samples from  Cedynia  and  Lednica  in  Poland , possibly Slavic, had been studied, the 855 sampled modern Bulgarians come out overally the closest group to these samples out of 20 other European nations and moreover, they share the highest value of haplotypes with the medieval Polish population more than any other compared nation does. Those medieval haplogroups included H, H1a, K1, K2, X2, X4, HV, J1b, R0a, HV0, H5a1a, N1b, T1a, J1b and W. The samples came out distant from modern Polish population, but nearest to the modern Bulgarian and Czech population. [ 73 ]  20 medieval(9-12th century) samples from Slovakian sites Nitra Šindolka and 8 from  Čakajovce  were compared to modern population and Bulgarians, and Portuguese came out nearest to them by genetic distance, however all these came out distant to modern Slovak population. [ 74 ]


Further evidence from  ancient DNA , reconsiderations of mutation rates, and collateral evidence from autosomal DNA growth rates suggest that the major period of European population expansion occurred after the Holocene. Thus the current geographic spread and frequency of haplogroups has been continually shaped from the time of Palaeolithic colonization to beyond the Neolithic. [ 75 ]  This process of genetic shaping continued into recorded history, such as the Slavic migrations. [ 76 ]


Recent studies of  ancient DNA  have revealed that European populations are largely descending from three ancestral groups. The first one are Paleolithic Siberians, the second one are Paleolithic European  hunter-gatherers , and the third one are early farmers and later arrivals from the Near East and West Asia. According to this, Bulgarians are predominantly (52%) descending from early  Neolithic farmers  spreading the agriculture from Anatolia, and from West Asian Bronze Age invaders and cluster together with other Southern Europeans. Another of the admixture signals in that farmers involves some ancestry related to East Asians, with ~ 2% total Bulgarian ancestry proportion linking to a presence of nomadic groups in Europe, from the time of the Huns to that of the Ottomans. A third signal involves admixture between the North European group from one side and the West Asian - Early farmers' group from another side, at approximately the same time as the East Asian admixture, ca. 850 AD. This event may correspond to the expansion of Slavic language speaking people. The analysis documents the hunter-gatherer admixture in Bulgarians at a level from ca. 1/3. [ 77 ]  The impact of  Yamnaya culture  is estimated at 20-30%, which is most common among the Slavs.


A later study published by  Cell Press  in 2023, focused on demographic developments in the Balkan peninsula during the first millennium CE, also examined the DNA of present-day populations from the Balkans, among them Bulgarians. It determined that present-day Bulgarians, like all other populations in the region, were affected by two major processes that took place following the end of the Iron Age:


Firstly, a 19.3±6.6% of their DNA could be traced to the Bulgarian Early Iron Age natives, another 23.8±7.6% from a Roman-era Balkan population with such Eastern Mediterranean ancestry, additionally, a minor contribution from a Bronze-to-Iron Age expansion in the Balkans, associated with Western Anatolia was given as contributing 5.7±2.9% of the present-day Bulgarian genome. Secondly, a demographic process which was found to have already been underway no later than 700 CE, and brought ancestry akin to that of present-day Eastern European  Slavic-speaking  populations contributed an even more significant component of the present-day Bulgarian genome, making up - according to the aforementioned model - 51.2±2.2% of present-day Bulgarian ancestry. The researchers also found the presence of two other waves of immigrants during the first half of the research period. One is from Central and Northern Europe, and the other from the steppe zone which stretches from the Northern Black Sea region to Kazakhstan. However, traces of these peoples are almost lost after 700 AD and they do not left a serious DNA mark among the Balkan populations of today. [ 78 ]


The  Bacho Kiro Cave  has yielded the oldest human remains ever to be found in Bulgaria which are unrelated to the modern-day Europeans. At one of the earliest known  Aurignacian  burials (layer 11), two pierced animal teeth were found and ordered into the distinct  Bachokiran artifact assemblage .  Radiocarbon dated  to over 43,000 years ago, they currently represent the oldest known ornaments in Europe. [ 79 ]  With an approximate age of 46,000 years, [ 80 ]  human fossils consist of a pair of fragmented  mandibles  including at least one  molar . Whether these early humans were in fact  Homo sapiens  or  Neanderthals  was disputed [ 81 ] [ 82 ]  until morphological analysis of a tooth and mitochondrial DNA of bone fragments established that remains were those of  Homo sapiens . In samples F6-620 and AA7-738 identified  mitochondrial haplogroup M , in samples WW7-240 and CC7-335 determined the  mitochondrial haplogroup N , in sample CC7-2289 identified  mitochondrial haplogroup R , in sample of BK-1653 identified  mitochondrial haplogroup U8 . [ 83 ] [ 84 ]


Three  Initial Upper Paleolithic  individuals (c. 44,000 to 40,000 years ago) from Bacho Kiro cave were each found to have relatively high levels of Neanderthal ancestry, with their genomes suggesting a recent Neanderthal ancestor in all three individuals perhaps six or seven generations back. In the single dispersal  Out of Africa theory , it is believed that populations related to the Initial Upper Palaeolithic population of Bacho Kiro cave contributed ancestry to later Asian populations, because of genetic similarity and to some early West Europeans such as the c. 35,000 year old individual from the  Goyet Caves , Belgium, known as 'GoyetQ116-1'. Populations related to these earlier individuals did not contribute detectable ancestry to later European populations. However in the multiple dispersal  Out of Africa theory , East Asians are found to have a more distant split time from East African populations (73-88kya) compared to modern Europeans (57-76 kya) [ 85 ]  which could mean that the Bacho Kiro remains could be from a migration of  anatomically modern humans  from Asia. [ 86 ] [ 87 ]  In 2022, a study determined that the IUP-affiliated Bacho Kiro remains were part of an  Initial Upper Paleolithic  wave (>45kya) "ascribed to a population movement with uniform genetic features and material culture" ( Ancient East Eurasians ), and sharing deep ancestry with other ancient specimens such as the  Ust'-Ishim man  and the  Tianyuan man , as well as ancestors of modern day  Papuans  (Australasians). The Bacho Kiro population associated with the IUP material culture in Europe went extinct and replaced by the later Upper Paleolithic migration associated with  West Eurasians  (represented by the  Kostenki-14  remains). [ 88 ]


Population genetics  is a scientific discipline which contributes to the examination of the human evolutionary and historical migrations. Particularly useful information is provided by the research of two uniparental markers within our genome, the  Y-chromosome  (Y-DNA) and  mitochondrial DNA  (mtDNA), as well as  autosomal DNA . The data from Y-DNA and autosomal DNA suggests that the  Croats  mostly are descendants of the  Slavs  of the medieval  migration period , according to mtDNA have genetic diversity which fits within a broader European maternal genetic landscape, and overall have a uniformity with other South Slavs from the territory of former  Yugoslavia .


There are many  Paleolithic  period sites located in the territory of Croatia, mostly ascribed to the  Mousterian  phase in the  Middle Paleolithic  period. In the Neolithic period in  Southeast Europe  were founded major cultures like  Vinča ,  Varna ,  Starčevo . In the  Bronze Age  happened symbiosis between  Proto-Indo-Europeans  of  Kurgan culture  and autochthonous populations, leading to the formation among others also of Proto- Illyrians . They gradually mixed and were assimilated by the  Romans ,  Celts ,  Ostrogoths , and finally  Early Slavs  since the late 6th century.


In the 2014 study, of the three successfully generated SNP profiles of Neolithic  Starčevo culture  samples from  Vinkovci , two belonged to Y-DNA haplogroup G2a-P15 and one to I2a1-P37.2, which could indicate G2a as potential representatives of the spread of farming from the Near East to Europe, while I2a as Mesolithic substratum in Europe. [ 1 ]  In the 2018 study, 10 out of 17 samples from Croatia had a successful Y-DNA sequencing; two Croatia  Cardial Neolithic  (6005–5786 BCE) samples from Zemunica Cave belonged to  C1a2  and E1b1b1a1b1, Early-Neolithic Starčevo culture (5837–5659 BCE) from  Beli Manastir -Popova zemlja to C, Early-Neolithic Croatia  Impressa culture  (5641–5560 BCE) from Kargadur to G2a2a1, two Middle-Neolithic  Sopot culture  (5207–4546 BCE) samples from  Osijek  to G2a2a1 and J2a1, Late-Neolithic Sopot (4790–4558 BCE) from Beli Manastir-Popova zemlja to I, two  Vučedol culture  (2884–2582 BCE) samples from Beli Manastir-Popova zemlja and Vučedol Tell to R1b1a1a2a2 and G2a2a1a2a, and the Early-Middle Bronze Age (1631–1521 BCE) sample from Veliki Vanik belonged to J2b2a. [ 2 ]


In 2019 were autosomally analyzed three 5th century individuals with  artificial cranial deformation  from Osijek, probably of Hunnic or Germanic origin. According to  Principal component analysis  and Admixture methods, one of them had East/Northeast Asian, another European/Caucasian and third Near Middle Eastern/North African ancestry. [ 3 ]


In the 2021, was made a genome-wide analysis on 38 out of 41 individuals from the 6200 years old massacre at village  Potočani  in Eastern Croatia. [ 4 ] [ 5 ]  They belonged to the Middle  Copper Age   Lasinja culture , and autosomally 70% of them were not in close kinship implying "a community composed of many family groups". According to Admixture and PCA it revealed "that the analyzed individuals are slightly shifted from the Anatolia Neolithic cluster in the direction of Western European hunter-gatherers, similar to other Middle to Late Neolithic European farmers before the arrival of  [Western] steppe  ancestry", as on average had approximately 91%  Anatolian Neolithic Farmers  (ANF) and 9%  Western Hunter-Gatherer  (WHG)-related ancestry, being very distant from modern Croats. Among them were present "paternal lineages typical of Balkan Neolithic populations (eleven G2a2, two I2a1a(xI2a1a2), one I2a2, C and C1a2-V20)". [ 6 ]


In the same year were analyzed additional 19 Middle Neolithic of Sopot culture, 1 Copper Age and 1 Roman age sample from Beli Manastir-Popova zemlja as well as 7 Bronze Age samples of  Transdanubian Encrusted Pottery culture  from  Jagodnjak -Krčevine in Eastern Croatia. According to Admixture, Croatian Neolithic samples have 97.6-100% ANF-ancestry, Copper Age samples have 71% ANF-ancestry and 29%  Western Steppe Herders  (WSH)-ancestry, while Bronze Age samples 47% ANF, 20% WHG and 33% WSG-ancestry. The observed Y-DNA haplogroups in Neolithic/Copper period were two G2a2a-PF3147, one G2a2b2a1a1-PF3345, one C1a2b-Z38888, two I2a2a-M223 and one J-M304; in Bronze period were only four G2a2a1a2a2a1-Z31430 and one G2a2a1-PF3148. The one Roman period individual from 260 to 402 CE, "who may or may not be representative for the wider population in that time period", had autosomal genetic combination very similar to present-day Southeastern Europeans (including Croats), "adding an important data point to the substantial shift in ancestry that occurred in the region between the Bronze Age and today". He carried  Scythian -Eurasian Y-DNA haplogroup  R1a1a1b2a2-Z2124  > R1a1a1b2a2b1-F1345 (R-F1019* subclade [ 7 ] ). [ 8 ]  Between 2018 and 2022 studies analyzed many samples related to the  Cetina culture , Proto- Illyrians  and specific Illyrian tribes ( Iapydes  and  Liburni ), finding that their paternal lineage almost exclusively belonged to the J2b-L283 haplogroup. [ 2 ] [ 9 ] [ 10 ]


A 2022  archaeogenetic  study published in  Science  compared ancient, medieval, and modern Balkans population samples and found that the  medieval Slavic migrations  "profoundly affected the region", resulting in the reduction of Anatolian Neolithic ancestry in Southeastern Europe. Pre-Slavic Balkan populations have the most of the Anatolian Neolithic component of ancestry, whereas present-day Slavs outside the Balkans have the least, "with present-day people from Southeastern Europe", including Croats, "intermediate between the two extremes", with Greeks and Albanians having the most while Croats and Hungarians the least. [ 11 ]  A 2023 archaeogenetic study published in  Cell  confirmed that the spread of Slavic language and identity was because of large movements of people of both males and females with specific Eastern European ancestry and that "more than half of the ancestry of most peoples in the Balkans today comes from the Slavic migrations, with around a third Slavic ancestry even in countries like Greece where no Slavic languages are spoken today". [ 12 ] [ 13 ]  The pre-Slavic period population of Croatia and the region had Y-DNA haplogroups E-V13, R1b, J2b, J2a, G2a while with Slavic migration arrived R1a-Z282 and I2a-L621 among others. [ 13 ]


Genetically , on the paternal  Y chromosome  line and studies published between 2003 and 2008, a majority (>75%) of male Croats from Croatia belongs to one of the three major European Y-DNA  haplogroups  –  I2  (32% [ 14 ] [ 15 ] [ 16 ] -34% [ 17 ] ),  R1a  (27% [ 17 ] -34% [ 14 ] [ 15 ] [ 16 ] ) and  R1b  (12% [ 17 ] -15% [ 14 ] [ 15 ] [ 16 ] ), while a minority (>25%) belongs to haplogroups  E  (9% [ 17 ] ),  I1  (5% [ 15 ] -9% [ 17 ] ),  J  (4% [ 17 ] ),  N  (2% [ 17 ] ), and  G  (1% [ 17 ] ). According to recent and more extensive studies published between 2012 and 2022, a majority (65%) of male Croats from Croatia belongs to haplogroups I2 (39%-40%) and R1a (22%-24%), while a minority (35%) belongs to haplogroups E (10%), R1b (6%-7%), J (6%-7%), I1 (5-8%), G (2%),  Q  (0-1.93%),  H  (0-1.8%),  T  (0.6%), N (0-0.6%) and  L  (0.2%). [ 18 ] [ 19 ]


Haplogroup I  among Croats from Croatia is around 43.5%, [ 18 ]  while in  Šokci , a subgroup of Croats from eastern Croatia, is between 44% and 50% of the same haplogroup. [ 20 ] [ 21 ]  It is divided in two major subdivisions – subclade  I2  (37.7%), typical for the populations of the Balkans and Eastern Europe, and  I1  (5.8%), typical for the populations of Scandinavia. [ 17 ]  From the I2 subclade, the most prevailing is I2a1a (I-P37.2) i.e. its subclade lineage I-M423 (18,400-14,000 YBP) > I-Y3104 (14,000-11,400 YBP) > I2a1a2b-L621 (11,400-6,500 YBP) > I-CTS10936 (6,500-5,600 YBP) > I-S19848 (5,600 YBP) > I-CTS4002 (5,600-5,100 YBP) > I-CTS10228 (5,100-3,400 YBP) > I-Y3120 (3,400-2,100 YBP) > I-S17250 (2,100-1,850 YBP) >  I-PH908  (1,850-1,700 YBP), [ 22 ]  which is typical of the South Slavic populations of  Southeastern Europe , being highest in  Bosnia-Herzegovina  (>50%). [ 16 ]  In Croatia the highest frequency is observed in  Dalmatia , peaking in cities of  Split  (50.48%),  Dubrovnik  (53%) and  Zadar  (60%), [ 23 ] [ 19 ]  as well southern Adriatic islands of  Vis  (44.6%), [ 23 ]   Brač  and  Korčula  (~55%), and  Hvar  (52.88-65%). [ 17 ] [ 19 ]  It is around average in the cities of  Pula  (31.31%) and  Osijek  (27.6-40.91%), [ 17 ] [ 19 ]  some southern  Mljet  (33.3%),  Lastovo  (33.3%) and middle Adriatic islands of  Pašman  (37.2%) and  Pag  (36.4%). [ 23 ]  The frequency is lowest in the town of  Varaždin  (18%) in northwestern Croatia, [ 19 ]  in the western mountainous  Žumberak  region (18.2%), [ 23 ]  and in the middle-northern islands of  Cres  (3%),  Dugi otok  (11.4%),  Ugljan  (24.6%) and  Krk  (27%). [ 14 ] [ 23 ]  The highest frequency is found in  Croats of Bosnia and Herzegovina  (63.8-73.3%). [ 16 ] [ 17 ]  Based on 8 STR marker genetic distances closest are populations of near countries, but also depending on method, Ukraine, Belarus, Poland and Russia. [ 24 ]  The subclade's I-P37.2 very high frequency in the Western Balkans diminishes in all directions. [ 25 ] [ 23 ]  The population with haplogroup I migrated to Europe from the Middle East, approximately 25,000-13,000 years ago. It represents the Paleolithic and Mesolithic  population of hunter gatherers  in Europe. However, in comparison to older research which argued a prehistoric autochthonous origin of the contemporary haplogroup I2 in Croatia and the Balkans, [ nb 1 ]  Battaglia  et al.  (2009) already observed highest variance of the haplogroup in  Ukraine , and Zupan  et al.  (2013) noted that it suggests it arrived with  Slavic migration  from the homeland which was in present-day Ukraine. [ 30 ]  The recent research by O.M. Utevska (2017), concluded that the haplogroup  STR   haplotypes  have the highest diversity in Ukraine, with ancestral STR marker result "DYS448=20" comprising " Dnieper - Carpathian " cluster, while younger derived result "DYS448=19" comprising the "Balkan cluster" which is predominant among the South Slavs. [ 31 ]  This "Balkan cluster" also has the highest variance in Ukraine, which indicates that the very high frequency in the Western Balkan is because of a  founder effect . [ 31 ]  Utevska calculated that the STR cluster divergence and its secondary expansion from the middle reaches of the Dnieper river or from  Eastern Carpathians  towards the Balkan peninsula happened approximately 2,860 ± 730 years ago, relating it to the times before Slavs, but much after the decline of the  Tripolye culture . [ 31 ]  However, STR-based calculations give overestimated dates, [ 32 ] [ 33 ]  and more specifically, the cluster is represented by a single SNP, I-PH908, known as I2a1a2b1a1a1c in  ISOGG  phylogenetic tree (2019), and according to YFull YTree it formed and had TMRCA approximately 1,850-1,700 YBP. [ 22 ]  Although it is dominant among the modern Slavic peoples on the territory of the former Balkan provinces of the  Roman Empire , until now it was not found among the samples from the Roman period and is almost absent in contemporary population of  Italy . [ 34 ]  According to Pamjav  et al.  (2019) and Fóthi  et al.  (2020), the distribution of ancestral subclades like of I-CTS10228 among contemporary carriers indicates a rapid expansion from Southeastern  Poland , is mainly related to the Slavs and their medieval migration, and the "largest demographic explosion occurred in the Balkans". [ 34 ] [ 35 ]  According to Olalde  et al.  (2023) it was confirmed that I2-L621 was absent before medieval period in the Balkans and arrived with the Slavs. [ 13 ]


R1a1a1-M17  (22.1%-25.6%) is the second  most prevailing haplogroup. [ 18 ]  The haplogroup R-M17 in Croatia is mostly divided into two subclades, R-M558 which is predominant (19.2%), and  R-M458  (4.9%), while  R-Z282  is rare (1.2%). [ 23 ]  It has highest frequency in northern (29.1%) and central (23.6%) region, and almost the same frequency in eastern (18.6%), southern (19.1%), and western (20%) region of Croatia. [ 18 ]  The highest local frequency of R1a1a1-M17 was observed in the Croats from Varaždin (38%) and Osijek (26-39%), [ 17 ] [ 19 ]  Žumberak (34.1), [ 23 ]  and in the middle-northern islands of Dugi Otok (34.1%), Krk (37%), Pašman (38%) and Cres (56.6%), [ 14 ] [ 23 ]  being similar to the values of the other Slavs, like Slovenes, Czechs and Slovaks. The frequency is lower in Šokci from eastern Croatia (16%), [ 20 ] [ 21 ]  in the city of Dubrovnik (13.4%) and Split (19%) in Dalmatia, as well on the southern islands of Hvar (8-10.58%) and Vis (17%). [ 14 ] [ 23 ] [ 19 ]  In Bosnian Croats, the frequency is similar to those of other South Slavs (12%). [ 17 ] [ 36 ]  Considering subclades, the only outlier is island of Cres which had almost equal percentage of R1a-M558 (29.3%) and R1a-M458 (27.3%). [ 23 ]  Based on 8 STR marker genetic distances closest are populations of near countries, but also depending on method, Belarus, Slovakia, Poland and Russia. [ 24 ]  The R-M558 subclade is more frequent among  East Slavs  in Eastern Europe and Volga-Ural region, while R-M458 among  West Slavs  in Central and Eastern Europe. Both are present in "informative frequencies in Balkan populations with known Slavonic heritage". [ 37 ]  R-M558 subclade CTS1211 was also found among Hungarian conquerors which indicates mixing and assimilation of the Slavs among the Hungarians. [ 38 ]


The highest frequency of the haplogroup  Haplogroup R1b  (7.9%-9.1%), which in Croatia is divided into several subclades (mainly  R-L23  and its subclade  R-U152 ), has in northern (10.9%) and central (11.8%) region of Croatia, [ 18 ]  while locally in the Croats from the island of Krk (16.2%) and  Dugi Otok  (25%), [ 14 ] [ 23 ]  and Žumberak (11.3%), [ 23 ]  while in the southern islands, city of Dubrovnik (3.9%) and in Bosnian Croats it is almost absent (1-6%), [ 14 ] [ 17 ] [ 23 ] [ 19 ]  or like in Osijek it was not found. [ 17 ]  These two haplogroups (R1a and R1b) are connected to  Proto-Indo-Europeans  migration from the Eurasian area some 5,000 years ago, [ 25 ]  with R1a particularly to Slavic population's migration. [ 16 ] [ 23 ]  Their frequency show north–south gradiation and an opposite frequency distribution to the haplogroup I-P37.2, [ 25 ]  and the highest frequency is observed in the northern, central and eastern Croatia. [ 14 ] [ 17 ] [ 18 ]


From the haplogroup E (9.8%-10.6%) among Croats the most frequent is subclade  E1b1b1a1b-V13  (6.7%), while E1b1b1a3-M149 and  E1b1b1c-M123  were also found in small numbers (1.1%). [ 17 ]   E-V13  it's typical of the populations of south-eastern Europe, peaking among Kosovo Albanians (44%), and is also high among the Macedonians, Greeks, Romanians, Bulgarians and Serbs. [ 16 ]  The highest frequency in Croatian mainland has been found in Varaždin (16%) and Žumberak (18.2%) [ 23 ] [ 19 ]  in central islands Dugi Otok (15.9%) and Ugljan (13.2%), as well southern islands  Vis  (23.4%) and  Mljet  (15.4%). [ 23 ]  In the northern islands of Cres (3%) [ 23 ]  and Krk (6.8%) was similar to other southern islands (3.7-4.3%). [ 14 ]  In Bosnian Croats the frequency was the same as among the Croats from Croatia (8.9%). [ 17 ]  Subclades of J1 are rare in Croatia, while J2 are higher in Croats from Croatia, peaking in Croats from Osijek (10.2%) and central islands Ugljan (10.2%) and Pašman (16.6%) as well the northern island of Krk (10.8%) and Cres (14.1%), [ 14 ] [ 17 ] [ 23 ]  than in Bosnian Croats (both 1.1%). [ 17 ]  Subclade G2a-P15 both in Croatian and Bosnian Croats is found in low numbers (1.1%), [ 17 ]  but peaks locally in the north-eastern town of Osijek (13.8%), [ 17 ]  and the southern islands of Mljet (15.4%), Korčula (10.4%), Brač (6%) as well northern island Cres (7%). [ 14 ] [ 23 ]  The haplogroup E and J are related to post-LGM, Neolithic migration of a population from Anatolia who brought with them domestication of wild animals and plants. Specifically, the haplogroup E's subclade probably arose locally in the Balkan not earlier than 8,000-10,000 years ago. These haplogroups show south–north gradiation. [ 25 ]  The haplogroup G could have been present in Europe during the LGM or population with some of its subclades arrived with early farmers. [ 25 ]


Haplogroup's  N  subclades are rare in Croatia (0-2.2%). [ 17 ] [ 23 ] [ 19 ]  It is very frequent in the  Far East , like Siberia and China, while in Europe in Finns (60%) and in the Baltic countries (45%). Unusually for European populations, another central Asian-Siberian haplogroup  Q  was found in unusually high frequencies due to founder effect in the islands of Hvar (7.69-14%), Lastovo (8.3%) and Korčula (6%). [ 14 ] [ 23 ] [ 19 ]  It was also found in an individual from Herzegovina. [ 16 ]  In later studies, a Croatian from Bosnia and Herzegovina was confirmed to belong to the subclade Q-L472>Z2902>B285>B29 found in Central-East Asia, [ 39 ]  while a Croatian from Croatia, another one from the island of Hvar, and a Croatian of Jewish origin in Poland belonged to the subclade Q-L245>Y2998>Y2209 which is mostly found in the Middle East and  Ashkenazi Jews . [ 40 ]


The region of modern-day Croatia was part of a wider Balkan region which may have served as one of several  refugia  during the LGM, a source region for the recolonization of Europe during the post-glacial period and Holocene (10,000 years ago). [ 17 ] [ 25 ]  The eastern Adriatic coast was much further south. [ 15 ]  The northern and the western parts of that sea were steppes and plains, while the modern Croatian islands (rich in Paleolithic archeological sites) were hills and mountains. [ 15 ] [ 17 ]  The region had a specific role in the structuring of European, and particularly among Slavic, paternal genetic heritage, characterized by the predominance of R1a and I, and scarcity of E lineages. [ 16 ]  The contemporary insular population's genetic diversity is characterized by strong isolation and  endogamy . [ 41 ]


In the table below is cited the most extensive study (2012) until now on the population in Croatia. It is a national reference  DNA database  of 17  loci  system which acquired Y- STR   haplotypes  were predicted in estimated (over 90% probability) Y- SNP   haplogroups . [ 18 ]  The sub-populations were divided in five regions which sub-populations showed strong similarity and homogeneity of paternal genetic contribution, all clustering together, with exception of sub-population from southern Croatia who showed a mild difference. In addition to high degree of overall homogeneity, there are gradient similarities to a central European cluster (Germanic, Slavic, Hungarian), and a southern European cluster (Bosnian, Serbian, Bulgarian, Macedonian, Romanian, Albanian, South Italian), going from north to south (and east to west). [ 18 ]  Out of the neighboring countries and ethnic peoples are closest to them Bosnia and Herzegovina and  Bosniaks . [ 42 ] [ 43 ] [ 44 ]