The simplest case of an overhang is a single nucleotide. This is most often  adenine  and is created as a 3' overhang by some  DNA polymerases . Most commonly this is used in cloning  PCR  products created by such an enzyme. The product is joined with a linear DNA molecule with a 3'  thymine  overhang. Since adenine and thymine form a  base pair , this facilitates the joining of the two molecules by a ligase, yielding a circular molecule. Here is an example of an A-overhang:


Longer overhangs are called  cohesive ends  or  sticky ends . [ 3 ]  They are most often created by  restriction endonucleases  when they cut DNA. Very often they cut the two DNA strands four base pairs from each other, creating a four-base 3' overhang in one molecule and a complementary 3' overhang in the other. These ends are called cohesive since they are easily joined back together by a ligase.


For example, these two "sticky" ends (four-base 5' overhangs) are compatible:


Also, since different restriction endonucleases usually create different overhangs, it is possible to create a plasmid by excising a piece of DNA (using a different enzyme for each end) and then joining it to another DNA molecule with ends trimmed by the same enzymes. Since the overhangs have to be complementary in order for the ligase to work, the two molecules can only join in one orientation. This is often highly desirable in  molecular biology .


Sticky ends can be converted to blunt ends by a process known as blunting, which involves filling in the sticky end with complementary nucleotides. This yields a blunt end, however, sticky ends are often preferable, meaning the main use of this method is to label DNA by using radiolabeled nucleotides to fill the gap. [ 4 ]  Blunt ends can also be converted to sticky ends by addition of double-stranded linker sequences containing recognition sequences for restriction endonucleases that create sticky ends and subsequent application of the restriction enzyme or by homopolymer tailing, which refers to extending the molecule's 3' ends with only one nucleotide, allowing for specific pairing with the matching nucleotide (e.g. poly-C with poly-G). [ 3 ]


Across from each single strand of DNA, we typically see  adenine  pair with  thymine , and  cytosine  pair with  guanine  to form a parallel complementary strand as described below.  Two nucleotide sequences which correspond to each other in this manner are referred to as complementary:


A frayed end refers to a region of a double stranded (or other multi-stranded) DNA molecule near the end with a significant proportion of non-complementary sequences; that is, a sequence where nucleotides on the adjacent strands do not match up correctly:


The term "frayed" is used because the incorrectly matched nucleotides tend to avoid bonding, thus appearing similar to the strands in a fraying piece of rope.


Although non-complementary sequences are also possible in the middle of double stranded DNA, mismatched regions away from the ends are not referred to as "frayed".


Ronald W. Davis  first discovered sticky ends as the product of the action of  EcoRI , the restriction  endonuclease . [ 5 ]


Sticky end links are different in their stability. Free energy of formation can be measured to estimate stability. Free energy approximations can be made for different sequences from data related to oligonucleotide UV thermal denaturation curves. [ 6 ]  Also predictions from molecular dynamics simulations show that some sticky end links are much stronger in stretch than the others. [ 7 ]


Systematic evolution of ligands by exponential enrichment  ( SELEX ), also referred to as  in vitro selection  or  in vitro evolution , is a  combinatorial chemistry  technique in  molecular biology  for producing  oligonucleotides  of either single-stranded  DNA  or  RNA  that specifically bind to a target  ligand  or ligands. These single-stranded  DNA  or  RNA  are commonly referred to as  aptamers . [ 1 ] [ 2 ] [ 3 ]
Although SELEX has emerged as the most commonly used name for the procedure, some researchers have referred to it as  SAAB  (selected and amplified binding site) and  CASTing  (cyclic amplification and selection of targets) [ 4 ] [ 5 ]  SELEX was first introduced in 1990. In 2015, a special issue was published in the  Journal of Molecular Evolution  in the honor of quarter century of the discovery of SELEX. [ 6 ]


The process begins with the synthesis of a very large oligonucleotide library, consisting of randomly generated sequences of fixed length flanked by constant  5'  and  3'  ends. The constant ends serve as  primers , while a small number of random regions are expected to bind specifically to the chosen target. For a randomly generated region of length  n , the number of possible sequences in the library using conventional DNA or RNA is 4 n  ( n  positions with four possibilities (A,T,C,G) at each position). The sequences in the library are exposed to the target ligand - which may be a  protein  or a  small organic compound  - and those that do not bind the target are removed, usually by  affinity chromatography  or target capture on paramagnetic beads. [ 7 ]  The bound sequences are eluted and amplified by  PCR [ 2 ] [ 3 ]  to prepare for subsequent rounds of selection in which the stringency of the elution conditions can be increased to identify the tightest-binding sequences. [ 2 ]  A caution to consider in this method is that the selection of extremely high, sub- nanomolar  binding affinity entities may not in fact improve specificity for the target molecule. [ 8 ]  Off-target binding to related molecules could have significant clinical effects.


SELEX has been used to develop a number of aptamers that bind targets interesting for both clinical and research purposes. [ 9 ]  Nucleotides with chemically modified sugars and bases have been incorporated into SELEX reactions to increase the chemical diversity at each base, expanding the possibilities for specific and sensitive binding, or increasing stability in  serum  or  in vivo . [ 9 ] [ 10 ]


Aptamers have emerged as a novel category in the field of bioreceptors due to their wide applications ranging from biosensing to therapeutics. Several variations of their screening process, called SELEX have been reported which can yield sequences with desired properties needed for their final use. [ 11 ]


The first step of SELEX involves the synthesis of fully or partially randomized oligonucleotide sequences of some length flanked by defined regions which allow PCR amplification of those randomized regions and, in the case of RNA SELEX, in vitro transcription of the randomized sequence. [ 2 ] [ 3 ] [ 12 ]  While Ellington and Szostak demonstrated that chemical synthesis is capable of generating ~10 15  unique sequences for oligonucleotide libraries in their 1990 paper on in vitro selection, [ 3 ]  they found that amplification of these synthesized oligonucleotides led to significant loss of pool diversity due to PCR bias and defects in synthesized fragments. [ 3 ]  The oligonucleotide pool is amplified and a sufficient amount of the initial library is added to the reaction so that there are numerous copies of each individual sequence to minimize the loss of potential binding sequences due to stochastic events. [ 3 ]  Before the library is introduced to target for incubation and selective retention, the sequence library must be converted to single stranded oligonucleotides to achieve structural conformations with target binding properties. [ 2 ] [ 3 ]


Immediately prior to target introduction, the single stranded oligonucleotide library is often heated and cooled slowly to renature oligonucleotides into thermodynamically stable secondary and tertiary structures. [ 3 ] [ 7 ]  Once prepared, the randomized library is incubated with immobilized target to allow oligonucleotide-target binding. There are several considerations for this target incubation step, including the target immobilization method and strategies for subsequent unbound oligonucleotide separation, incubation time and temperature, incubation buffer conditions, and target versus oligonucleotide concentrations. Examples of target immobilization methods include  affinity chromatography  columns, [ 3 ]  nitrocellulose  binding assay filters , [ 2 ]  and  paramagnetic beads. [ 7 ]  Recently, SELEX reactions have been developed where the target is whole cells, which are expanded near complete  confluence  and incubated with the oligonucleotide library on culture plates. [ 13 ]  Incubation buffer conditions are altered based on the intended target and desired function of the selected aptamer. For example, in the case of negatively charged small molecules and proteins, high salt buffers are used for  charge screening  to allow nucleotides to approach the target and increase the chance of a specific binding event. [ 3 ]  Alternatively, if the desired aptamer function is in vivo protein or whole cell binding for potential therapeutic or diagnostic application, incubation buffer conditions similar to in vivo plasma salt concentrations and homeostatic temperatures are more likely to generate aptamers that can bind in vivo. Another consideration in incubation buffer conditions is non-specific competitors. If there is a high likelihood of non-specific oligonucleotide retention in the reaction conditions, non specific competitors, which are small molecules or polymers other than the SELEX library that have similar physical properties to the library oligonucleotides, can be used to occupy these non-specific binding sites. [ 13 ]  Varying the relative concentration of target and oligonucleotides can also affect properties of the selected aptamers. If a good  binding affinity  for the selected aptamer is not a concern, then an excess of target can be used to increase the probability that at least some sequences will bind during incubation and be retained. However, this provides no selective pressure for high  binding affinity , which requires the oligonucleotide library to be in excess so that there is competition between unique sequences for available specific binding sites. [ 2 ]


Once the oligonucleotide library has been incubated with target for sufficient time, unbound oligonucleotides are washed away from immobilized target, often using the incubation buffer so that specifically bound oligonucleotides are retained. [ 3 ]  With unbound sequences washed away, the specifically bound sequences are then eluted by creating denaturing conditions that promote oligonucleotide unfolding or loss of binding conformation including flowing in deionized water, [ 3 ]  using denaturing solutions containing urea and EDTA, [ 13 ] [ 14 ]  or by applying high heat and physical force. [ 7 ]  Upon elution of bound sequences, the retained oligonucleotides are  reverse-transcribed  to DNA in the case of RNA or modified base selections, [ 2 ] [ 3 ] [ 13 ]  or simply collected for amplification in the case of DNA SELEX. [ 15 ]  These DNA templates from eluted sequences are then amplified via  PCR  and converted to single stranded DNA, RNA, or modified base oligonucleotides, which are used as the initial input for the next round of selection. [ 2 ] [ 3 ]


One of the most critical steps in the SELEX procedure is obtaining single stranded DNA (ssDNA) after the PCR amplification step. This will serve as input for the next cycle so it is of vital importance that all the DNA is single stranded and as little as possible is lost. Because of the relative simplicity, one of the most used methods is using biotinylated reverse primers in the amplification step, after which the complementary strands can be bound to a resin followed by elution of the other strand with lye. Another method is asymmetric PCR, where the amplification step is performed with an excess of forward primer and very little reverse primer, which leads to the production of more of the desired strand. A drawback of this method is that the product should be purified from double stranded DNA (dsDNA) and other left-over material from the PCR reaction. Enzymatic degradation of the unwanted strand can be performed by tagging this strand using a phosphate-probed primer, as it is recognized by enzymes such as  Lambda exonuclease . These enzymes then selectively degrade the phosphate tagged strand leaving the complementary strand intact. All of these methods recover approximately 50 to 70% of the DNA. For a detailed comparison refer to the article by Svobodová et al. where these, and other, methods are experimentally compared. [ 16 ]  In classical SELEX, the process of randomized single stranded library generation, target incubation, and binding sequence elution and amplification described above are repeated until the vast majority of the retained pool consists of target binding sequences, [ 2 ] [ 3 ]  though there are modifications and additions to the procedure that are often used, which are discussed below.


In order to increase the specificity of aptamers selected by a given SELEX procedure, a negative selection, or counter selection, step can be added prior to or immediately following target incubation. To eliminate sequences with affinity for target immobilization matrix components from the pool, negative selection can be used where the library is incubated with target immobilization matrix components and unbound sequences are retained. [ 14 ] [ 17 ] [ 15 ]  Negative selection can also be used to eliminate sequences that bind target-like molecules or cells by incubating the oligonucleotide library with small molecule target analogs, undesired cell types, or non-target proteins and retaining the unbound sequences. [ 13 ] [ 15 ] [ 18 ]


To track the progress of a SELEX reaction, the number of target bound molecules, which is equivalent to the number of oligonucleotides eluted, can be compared to the estimated total input of oligonucleotides following elution at each round. [ 3 ] [ 19 ]  The number of eluted oligonucleotides can be estimated through elution concentration estimations via 260 nm wavelength absorbance [ 19 ]  or fluorescent labeling of oligonucleotides. [ 7 ]  As the SELEX reaction approaches completion, the fraction of the oligonucleotide library that binds target approaches 100%, such that the number of eluted molecules approaches the total oligonucleotide input estimate, but may converge at a lower number. [ 3 ]


Some SELEX reactions can generate probes that are dependent on primer binding regions for secondary structure formation. [ 7 ]  There are aptamer applications for which a short sequence, and thus primer truncation, is desirable. [ 20 ]  An advancement on the original method allows an RNA library to omit the constant primer regions, which can be difficult to remove after the selection process because they stabilize  secondary structures  that are unstable when formed by the random region alone. [ 21 ]


Recently, SELEX has expanded to include the use of chemically modified nucleotides. These chemically modified oligonucleotides offer many potential advantages for selected aptamers including greater stability and nuclease resistance, enhanced binding for select targets, expanded physical properties - like increased hydrophobicity, and more diverse structural conformations. [ 9 ] [ 10 ] [ 22 ]


The genetic alphabet, and thus possible aptamers, is also expanded using unnatural base pairs [ 23 ] [ 24 ]  the use of these unnatural base pairs was applied to SELEX and high affinity DNA aptamers were generated. [ 25 ]


FRELEX was developed in 2016 by  NeoVentures Biotechnology Inc  to allow the selection of aptamers without immobilizing the target or the oligonucleotide library. [ 26 ]  Immobilization is a necessary component of SELEX; however, it has the potential to inhibit key  epitopes , and thus weaken the likelihood of successful binding, particularly when working with small molecules. [ 27 ] [ 28 ]  FRELEX follows a similar overall methodology to SELEX; however, instead of immobilizing the target, the researcher introduces a series of random and blocker oligonucleotides to an immobilization field before introduction to the target. [ 26 ]  This allows the researcher to better target small molecules that may be lost during partitioning. [ 26 ]  It also can be used in some circumstances to select an aptamer library without knowing the target. [ 29 ]


Most modern aptamer selection methods strive to improve the conventional SELEX aptamer search method. [ 30 ]  Despite the publication of various methods aimed at increasing the affinity and specificity of aptamers, [ 31 ] [ 32 ] [ 33 ]  experimental approaches face limitations in the number and variety of sequences that can be examined and selected. Library capacity for SELEX experiments is practically limited to 10 15  candidates, whereas, assuming there is a 4-monomeric repertoire from which pools can be created, there are ~1.6 × 10 60  unique sequences in sequence space limited to a 100-residue matrix, which is clearly beyond experimental capabilities. [ 34 ]  The library of oligonucleotides must be extremely diverse and not contain linear, incapable of providing a stable spatial arrangement, and double-stranded structures; due to these limitations, oligonucleotide libraries can cover the diversity of only ~10 6  sequences. [ 35 ]  This means that existing aptamers may not fully cover the diversity of target molecules or may not have optimal properties due to limitations of the underlying method. To yield the best possible aptamers one must maximize the effectiveness of the discovery process and the library itself.


RNA and DNA secondary structure prediction by dynamic programming algorithms such as RNAfold ( ViennaRNA )  [ 36 ]  and by machine learning models such as SPOT-RNA, [ 37 ]  MXfold2  [ 38 ]  provides the opportunity to assess the ability of sequences in the primary library to fold into complex structures, allowing for the selection of only the most promising sequences from the entire pool. However, these algorithms are low-performance, making them poorly suited for this task. For this reason, algorithms like Ufold from the University of California  [ 39 ]  and AliNA from Xelari Inc.  [ 40 ]  have been developed, which demonstrate a significant increase in computational speed due to their faster architecture, and can be applied for preliminary  in silico  analysis of these libraries.


The technique has been used to evolve  aptamers  of extremely high binding affinity to a variety of target ligands, including  small molecules  such as  ATP [ 41 ]  and  adenosine [ 12 ] [ 42 ]  and proteins such as  prions [ 43 ]  and  vascular endothelial growth factor  (VEGF). [ 44 ]  Moreover, SELEX has been used to select high-affinity aptamers for complex targets such as tumor cells, [ 45 ] [ 46 ]  tumor exosomes, [ 47 ] [ 48 ]  or tumor tissue. [ 49 ]  Clinical uses of the technique are suggested by aptamers that bind  tumor markers , [ 50 ]   GFP -related  fluorophores , [ 51 ]  and a VEGF-binding aptamer trade-named  Macugen  has been approved by the FDA for treatment of  macular degeneration . [ 44 ] [ 52 ]  Additionally, SELEX has been utilized to obtain highly specific catalytic DNA or DNAzymes. Several metal-specific DNAzymes have been reported including the GR-5 DNAzyme (lead-specific), [ 53 ]  the CA1-3 DNAzymes (copper-specific), [ 54 ]  the 39E DNAzyme (uranyl-specific)  [ 55 ]  and the NaA43 DNAzyme (sodium-specific). [ 56 ]


These developed aptamers have seen diverse application in therapies for macular degeneration [ 52 ]  and various research applications including  biosensors , [ 20 ]  fluorescent labeling of proteins [ 57 ]  and cells, [ 58 ]  and selective enzyme inhibition. [ 59 ]


TILLING  ( Targeting Induced Local Lesions in Genomes ) is a method in  molecular biology  that allows directed identification of  mutations  in a specific  gene . TILLING was introduced in 2000, using the model plant  Arabidopsis thaliana , and expanded on into other uses and methodologies by a small group of scientists including  Luca Comai . TILLING has since been used as a  reverse genetics  method in other organisms such as  zebrafish ,  maize ,  wheat ,  rice ,  soybean ,  tomato  and  lettuce .


The method combines a standard and efficient technique of  mutagenesis  using a chemical mutagen such as  ethyl methanesulfonate  (EMS) with a sensitive DNA screening-technique that identifies single base mutations (also called point mutations) in a target gene. The TILLING method relies on the formation of  DNA   heteroduplexes  that are formed when multiple alleles are amplified by  PCR  and are then heated and slowly cooled.  A “ bubble ” forms at the mismatch of the two DNA strands, which is then cleaved by a single stranded  nuclease .  The products are then separated by size on several different platforms (see below).


Mismatches may be due to induced mutation, heterozygosity within an individual, or natural variation between individuals.


EcoTILLING  [ 1 ] [ 2 ] [ 3 ] [ 4 ]  is a method that uses TILLING techniques to look for natural mutations in individuals, usually for population genetics analysis.  DEcoTILLING  [ 5 ]  is a modification of TILLING and EcoTILLING which uses an inexpensive method to identify fragments. Since the advent of  NGS sequencing  technologies, TILLING-by-sequencing [ 6 ]  has been developed based on Illumina sequencing of target genes amplified from multidimensionally pooled templates to identify possible single-nucleotide changes.


There are several sources for single strand nucleases.  The first widely used enzyme was  mung bean nuclease , but this nuclease has been shown to have high non-specific activity, and only works at low pH, which can degrade PCR products and dye-labeled primers.  The original source for single strand nuclease was from CEL1, or CJE (celery juice extract), but other products have entered the market including Frontier Genomics’ SNiPerase enzymes, which have been optimized for use on platforms that use labeled and unlabeled PCR products (see next section).  Transgenomic isolated the single strand nuclease protein and sells it as a recombinant form.  The advantage of the recombinant form is that unlike the enzyme mixtures, it does not contain non-specific nuclease activity, which can degrade the dyes on the PCR primers.  The disadvantage is a substantially higher cost.


The first paper describing TILLING used  HPLC  to identify mutations (McCallum et al., 2000a).  The method was made more high throughput by using the  restriction enzyme  Cel-I combined with the LICOR gel based system to identify mutations (Colbert et al., 2001).  Advantages to using this system are that mutation sites can be easily confirmed and differentiated from noise.  This is because different colored dyes can be used for the forward and reverse primers.  Once the cleavage products have been run on a gel, it can be viewed in separate channels, and much like an RFLP, the fragment sizes within a lane in each channel should add up to the full length product size.  Advantages to the LICOR system are separation of large fragments (~ 2kb), high sample throughput (96 samples loaded on paper combs), and freeware to identify the mutations (GelBuddy).  Drawbacks to the LICOR system is having to pour slab gels and long run times (~4 hours).  TILLING and EcoTILLING methods are now being used on capillary systems from, Advanced Analytical Technologies, ABI and Beckman.


Several systems can be used to separate PCR products that are not labeled with dyes.  Simple agarose electrophoresis systems will separate cleavage products inexpensively and with standard lab equipment.  This was used to discover SNPs in chum salmon and was referred to as DEcoTILLING.  The disadvantage of this system is reduced resolution compared to polyacrylamide systems.  Elchrom Scientific sells Spreadex gels which are precast, can be high throughput and are more sensitive than standard polyacrylamide gels.  Advanced Analytical Technologies Inc sells the AdvanCE FS96 dsDNA Fluorescent System which is a 96 capillary electrophoresis system that has several advantages over traditional methods; including ability to separate large fragments (up to 40kb), no desalting or precipitation step required, short run times (~30 minutes), sensitivity to 5pg/ul and no need for fluorescent labeled primers.


Several TILLING centers exist over the world that focus on agriculturally important species:


TSE  or  Tris/Saline/EDTA , is a  buffer solution  containing a mixture of  Tris base ,  Sodium chloride  and  EDTA .


In molecular biology, TSE buffers are often used in procedures involving  nucleic acids . Tris-acid solutions are effective buffers for slightly basic conditions, which keep DNA deprotonated and soluble in water. The concentration of tris in the solution is kept near 25  mM . EDTA is a  chelator  of divalent  cations , particularly of  magnesium  (Mg 2+ ). As these ions are necessary co-factors for many enzymes, including contaminant  nucleases , the role of the EDTA is to protect the nucleic acids against enzymatic degradation. But since Mg 2+  is also a co-factor for many useful DNA-modifying enzymes such as  restriction enzymes  and  DNA polymerases , its concentration in TSE buffers is generally kept low (typically at around 2.5  mM ). The sodium chloride is generally kept at a concentration of 0.05  M . [ 1 ]


xDNA  (also known as  expanded DNA  or  benzo-homologated DNA ) is a size-expanded  nucleotide  system synthesized from the fusion of a  benzene ring  and one of the four natural bases:  adenine ,  guanine ,  cytosine , and  thymine . [ 1 ]  This size expansion produces an 8 letter alphabet which has a larger  information storage capacity  than natural  DNA's  (often referred to as B-DNA in literature) 4 letter alphabet. [ 2 ]  As with normal  base-pairing , A pairs with xT, C pairs with xG, G pairs with xC, and T pairs with xA. The  double helix  is thus 2.4 Å  wider than a natural double helix. [ 3 ] [ 4 ]  While similar in structure to B-DNA, xDNA has unique absorption, fluorescence, and stacking properties. [ 5 ] [ 6 ] [ 7 ]


Initially synthesized as an  enzyme  probe by Nelson J. Leonard's group, benzo-homologated adenine was the first base synthesized. Later,  Eric T. Kool 's group finished synthesizing the remaining three expanded  bases , eventually followed by yDNA ("wide" DNA), another benzo-homologated nucleotide system, and  naphtho -homologated xxDNA and yyDNA. xDNA is more stable when compared to regular DNA when subjected to higher temperature, and while entire strands of xDNA, yDNA, xxDNA and yyDNA exist, they are currently difficult to synthesize and maintain. Experiments with xDNA provide new insight into the behavior of natural B-DNA. The extended bases xA, xC, xG, and xT are naturally  fluorescent , and single strands composed of only extended bases can recognize and bind to single strands of natural DNA, making them useful tools for studying biological systems. [ 3 ] [ 8 ]  xDNA is most commonly formed with base pairs between a natural and expanded  nucleobase , however x-nucleobases can also be paired together. [ 5 ]  Current research supports xDNA as a viable genetic encoding system in the near future. [ 4 ]


The first  nucleotide  to be expanded was the  purine   adenine . Nelson J. Leonard and colleagues synthesized this original x-nucleotide, which was referred to as "expanded adenine". xA was used as a probe in the investigation of  active sites  of  ATP -dependent  enzymes , more specifically what modifications the  substrate  could take while still being functional. [ 8 ] [ 9 ]  Almost two decades later, the other three bases were successfully expanded and later integrated into a  double helix  by  Eric T. Kool  and colleagues. Their goal was to create a synthetic genetic system which mimics and surpasses the functions of the natural genetic system, [ 10 ]  and to broaden the applications of  DNA  both in living cells and in experimental  biochemistry . Once the expanded base set was created, the goal shifted to identifying or developing faithful replication enzymes and further optimizing the expanded DNA alphabet. [ 8 ]


In benzo-homologated purines (xA and xG), the  benzene ring  is bound to the  nitrogenous base  through nitrogen-carbon (N-C) bonds. Benzo-homologated pyrimidines are formed through carbon-carbon (C-C) bonds between the base and the benzene. [ 3 ]  Thus far, x-nucleobases have been added to strands of DNA using  phosphoramidite  derivatives, as traditional  polymerases  have been unsuccessful in synthesizing strands of xDNA. X-nucleotides are poor candidates as substrates for B-DNA polymerases as their size interferes with binding at the  catalytic domain . Attempts at using  template-independent  enzymes have been successful as they have a reduced geometric constraint for substrates.  Terminal deoxynucleotidyl transferase  (TdT) has been used previously to synthesize strands of bases which have been bound to  fluorophores . Using  TdT , up to 30  monomers  can be combined to form a double-helix of xDNA, however this  oligomeric  xDNA appears to inhibit its own extension beyond this length due to the overwhelming hydrogen bonding. In order to minimize inhibition, xDNA can be hybridized into a regular helix. [ 7 ] [ 11 ]


For xDNA to be used as a substitute structure for information storage, it requires a reliable replication mechanism. Research into xDNA replication using a  Klenow fragment  from  DNA polymerase I  shows that a natural base partner is selectively added in instances of single-nucleotide insertion. However,  DNA polymerase IV  (Dpo4) has been able to successfully use xDNA for these types of insertions with high fidelity, making it a promising candidate for future research in extending replicates of xDNA. [ 4 ]  xDNA's mismatch sensitivity is similar to that of  B-DNA . [ 2 ]


Similar to natural bases, x-nucleotides selectively assemble into a duplex-structure resembling B-DNA. [ 4 ]  xDNA was originally synthesized by incorporating a benzene ring into the nitrogenous base. However, other expanded bases have been able to incorporate  thiophene  and  benzo[b]thiophene  as well. xDNA and yDNA use benzene rings to widen the bases and are thus termed "benzo-homologated". Another form of expanded nucleobases known as yyDNA incorporate  naphthalene  into the base and are "naptho-homologated". xDNA has a rise of 3.2 Å  and a twist of 32°, significantly smaller than B-DNA, which has a rise of 3.3 Å  and a twist of 34.2° [ 3 ]  xDNA nucleotides can occur on both strands—either alone (known as "doubly expanded DNA" [ 8 ] ) or mixed with natural bases—or exclusively on one strand or the other. Similar to B-DNA, xDNA can recognize and bind complementary single-stranded  DNA  or  RNA  sequences. [ 2 ]


Duplexes formed from xDNA are similar to  natural duplexes  aside from the distance between the two sugar-phosphate backbones. xDNA helices have a greater number of base pairs per turn of the helix as a result of a reduced distance between neighbour nucleotides. NMR spectra report that xDNA helices are anti-parallel,  right-handed  and take an  anti  conformation  around the  glycosidic bond , with a C2'-endo sugar pucker. [ 5 ] [ 11 ]  Helices created from xDNA are more likely to take a B-helix over an A-helix conformation, [ 2 ]  and have an increased major groove width by 6.5 Å  (where the backbones are farthest apart) and decreased minor groove width by 5.5 Å  (where the backbones are closest together) compared to  B-DNA . Altering groove width affects the xDNA's ability to associate with  DNA-binding proteins , [ 12 ]  but as long as the expanded  nucleotides  are exclusive to one strand, recognition sites are sufficiently similar to  B-DNA  to allow bonding of  transcription factors  and small  polyamide  molecules. Mixed helices present the possibility of recognizing the four expanded bases using other DNA-binding molecules. [ 11 ]


Expanded nucleotides and their oligomeric helices share many properties with their natural  B-DNA  counterparts, including their pairing preference:  A  with  T ,  C  with  G . [ 11 ]  The various differences in chemical properties between xDNA and  B-DNA  support the hypothesis that the  benzene ring  which expands x-nucleobases is not, in fact, chemically inert. [ 5 ]  xDNA is more  hydrophobic  than  B-DNA , [ 7 ]  and also has a smaller  HOMO-LUMO  gap (distance between the highest occupied molecular orbital and lowest unoccupied molecular orbital) as a result of modified  saturation . [ 3 ]  xDNA has higher melting temperatures than  B-DNA  (a mixed decamer of xA and T has a melting temperature of 55.6 °C, 34.3 °C higher than the same decamer of A and T [ 11 ] ), and exhibits an "all-or-nothing" melting behaviour. [ 2 ]


Under lab conditions, xDNA orients itself in the  syn  conformation . This unfortunately does not expose the binding face of the xDNA nucleotides to face the neighbouring strand for binding, meaning that extra measures must be applied to alter the conformation of xDNA before attempting to form helices. However, the  anti  and  syn  orientations are practically identical energetically in expanded bases. [ 9 ]  This conformational preference is seen primarily in  pyrimidines , and  purines  display minimal preference for orientation. [ 5 ]


Stacking of the  nucleotides  in a  double helix  is a major determinant of the helix's stability. With the added  surface area  and  hydrogen  available for bonding, stacking potential for the nucleobases increases with the addition of a  benzene  spacer. By increasing the separation between the  nitrogenous bases  and either sugar-phosphate backbone, the helix's stacking energy is less variable and therefore more stable. The energies for natural nucleobase pairs vary from 18 to 52 kJ/mol. This variance is only 14–40 kJ/mol for xDNA. [ 8 ]


Due to an increased overlap between and expanded strand of  DNA  and its neighbouring strand, there are greater interstrand interactions in expanded and mixed helices, resulting in a significant increase in the helix's stability. xDNA has enhanced stacking abilities resultant from changes in inter- and intrastrand  hydrogen bonding  that arise from the addition of a  benzene  spacer, but expanding the bases does not alter hydrogen's contribution to the stability of the duplex. These stacking abilities are exploited by helices consisting of both xDNA and  B-DNA  in order to optimize the strength of the helix. Increased stacking is seen most prominently in strands consisting only of  A  and xA and  T  and xT, as  T -xA has stronger stacking interactions than  T - A . [ 3 ]


The energy resultant from  pyrimidines  ranges from 30 to 49 kJ/mol. The range for  purines  is between 40-58kJ/mol. By replacing one nucleotide in a double-helix with an expanded nucleotide, the strength of the stacking interactions increases by 50%. Expanding both nucleotides results in a 90% increase in stacking strength. While xG has an overall negative effect on the binding strength of the helix, the other three expanded bases outweigh this with their positive effects. The change in energy caused by expanding the bases is mostly dependent on the  rotation of the bond  about the nucleobases'  centers of mass , and center of mass stacking interactions improve the stacking potential of the helix. [ 5 ]  Because the size-expanded bases widen the helix, it is more thermally stable with a higher melting temperature. [ 7 ]


The addition of a  benzene  spacer in x- nucleobases  affects the bases'  optical absorption  spectra.  Time-dependent density functional theory  (TDDFT) applied to xDNA revealed that the benzene component of the highest occupied molecular orbitals ( HOMO ) in the x-bases pins the absorption onset at an earlier point than natural  bases . Another unusual feature of xDNA absorption spectra is the red-shifted  excimers  of xA in the low range. In terms of stacking fingerprints, there is a more pronounced hypochromicity seen in consecutive xA- T   base pairs .


Implications of xDNA's altered absorption include applications in  nanoelectronic technology  and  nanobiotechnology . The reduced spacing between x-nucleotides makes the  helix  stiffer, thus it is not as easily affected by  substrate ,  electrode , and functional  nanoparticle  forces. Other alterations to natural  nucleotides  resulting in different absorption spectra will broaden these applications in the future. [ 6 ]


One unique property of xDNA is its inherent  fluorescence . Natural bases can be bound directly to  fluorophores  for use in  microarrays ,  in situ  hybridization , and  polymorphism  analysis. However, these fluorescent natural bases often fail as a result of  self-quenching , which diminishes their fluorescent intensity and reduces their applicability as visual DNA tags. The  pi interactions  between the rings in x-nucleobases result in an inherent  fluorescence  in the violet-blue range, with a  Stokes shift  between 50 and 80 nm. They also have a  quantum yield  in the range of 0.3–0.6. xC has the greatest fluorescent emission. [ 10 ] [ 7 ]


After the creation of and successful research surrounding xDNA, more forms of expanded nucleotides were investigated. yDNA is a second, similar system of nucleotides which uses a  benzene ring  to expand the four  natural bases . xxDNA and yyDNA use  naphthalene , a polycyclic molecule consisting of two  hydrocarbon  rings. The two rings expand the base even wider, further altering its chemical properties.


The success and implications of xDNA prompted research to examine other factors which could alter  B-DNA 's chemical properties and create a new system for information storage with broader applications. yDNA also uses a  benzene ring , similar to xDNA, with the only difference being the site of addition of the  aromatic ring . The location of the  benzene ring  changes the preferred structure of the expanded helix. The altered conformation makes yDNA more similar to  B-DNA  in its orientation by changing the interstrand  hydrogen bonds . Stability is highly dependent on the bases' rotation about the link between the base and the sugar of the backbone. yDNA's altered preference for this orientation makes it more stable overall than xDNA. The location of the  benzene  spacer also affects the bases' groove geometry, altering neighbour interactions. The base pairs between y-nucleotides and natural nucleotides is planar, rather than slightly twisted as with xDNA. This decreases the rise of the  helix  even further than achieved by xDNA.


While xDNA and yDNA are quite similar in most properties, including their increased stacking interactions, yDNA shows superior mismatch recognition. y-pyrimidines display slightly stronger stacking interactions than x-pyrimidines as a result of the distance between the two  anomeric  carbons, which is slightly larger in yDNA. xDNA still has stronger stacking interactions in model helices, but adding either x- or y-pyrimidines to a natural  double helix  strengthens the intra- and interstrand interactions, increasing overall helix stability. In the end, which of the two has the strongest overall stacking interactions is dependent on the  sequence ; xT and yT bind  A  with similar strength, but the stacking energy of yC bound to  G  is stronger than xC by 4kJ/mol. yDNA and other expanded bases are part of a very young field which is highly understudied. Research suggest that the ideal conformation still remains to be discovered, but knowing that the  benzene  location affects the orientation and structure of expanded nucleobases adds information to their future design. [ 8 ]


Doubly-expanded (or  naphtho-homologated ) nucleobases incorporate a  naphthalene  spacer instead of a  benzene ring , widening the base twice as much with its two-ringed structure. These structures (known as xxDNA and yyDNA) are 4.8 Å  wider than  natural bases  and were once again created as a result of Leonard's research on expanded  adenine  in  ATP -dependent  enzymes  in 1984. No literature was published on these doubly-expanded bases for nearly three decades until 2013 when the first xxG was produced by Sharma, Lait, and Wetmore and incorporated along with xxA into a  natural helix . Although very little research has been performed on xxDNA, xx- purine  neighbours have already been shown to increase intrastrand stacking energy by up to 119% (as opposed to 62% in x-purines). xx- purine  and  pyrimidine  interactions show an overall decrease in stacking energies, but the overall stability of  duplexes  including pyrimidines and xx-purines increases by 22%, more than twofold that of pyrimidines and x-purines. [ 9 ]


xDNA has many applications in chemical and biological research, including expanding upon applications of natural  DNA , such as scaffolding. In order to create self-assembling nanostructures, a scaffold is needed as a sort of  trellis  to support the growth. DNA has been used as a means to this end in the past, but expanded scaffolds make larger scaffolds for more complex self-assembly an option. [ 1 ]  xDNA's  electrical conduction  properties also make it a prime candidate as a  molecular wire , as its  π-π interactions  help it efficiently conduct electricity. [ 3 ]  Its 8-letter alphabet ( A ,  T ,  C ,  G , xA, xT, xC, xG) gives it the potential to store 2 n  times more states per  sequence  than DNA, where  n  is the number of bases in the sequence. For example, combining 6 nucleotides of with  B-DNA  yields 4096 possible sequences, whereas a combination of the same number of nucleotides created with xDNA yields 262,144 possible sequences. Additionally, xDNA can be used as a fluorescent probe at enzyme  active sites , as was its original application by Leonard et al. [ 2 ]


xDNA has also been applied to the study of  protein-DNA interactions . Due to xDNA's natural  fluorescing  properties, it can easily be visualized in both lab and living conditions. [ 5 ]  xDNA is becoming more easy to create and  oligomerize , and its high-affinity binding to  complementary   DNA  and  RNA  sequences means that it can not only help locate these sequences floating around in the cell, but also when they are already interacting with other structures within the cell. [ 10 ]  xDNA also has potential applications in assays that employ  TdT  as it may improve reporters, and can be used as an  affinity tag  for interstrand bonding. [ 7 ]


The  Elston–Stewart algorithm  is an algorithm for computing the  likelihood  of observed  data on a  pedigree  assuming a general model under which specific genetic segregation, linkage and association models can be tested.  It is due to  Robert Elston  and  John Stewart .  It can handle relatively large pedigrees providing they are (almost) outbred.  When used for linkage analysis its computation time is exponential in the number of markers, in contrast to the  Lander-Green algorithm , which has computational time exponential in the number of pedigree members.


In  statistical genetics ,  Haseman–Elston   (HE) regression  is a  statistical regression  originally proposed for  linkage analysis  of  quantitative traits  in  sibling pairs . It was first developed by Joseph K. Haseman and  Robert C. Elston  in 1972. [ 1 ]  A much earlier implementation of sib-pair linkage analysis was proposed by  Lionel S. Penrose  in 1935 and 1938. [ 2 ] [ 3 ]  Notably, Penrose was the father of Nobel laureate theoretical physicist  Roger Penrose .


In 2000, Elston et al. and colleagues introduced a "revisited" and extended form of Haseman–Elston regression. [ 4 ]  Since then, further extensions of this revised model have been proposed. [ 5 ] [ 6 ] [ 7 ]  Despite its historical significance, HE regression has been described as "...a rusty weapon in the  genomics  analysis armory of the  GWAS  era." This criticism arises because HE regression relies on relatedness measured by identity by descent ( IBD ), rather than  identity by state  (IBS). However, adaptations of HE regression have been developed for association analysis in unrelated samples, where relatedness is measured by IBS. [ 8 ]


The  Lander–Green algorithm  is an algorithm, due to  Eric Lander  and  Philip Green  for computing the  likelihood  of observed genotype data given a  pedigree . It is appropriate for relatively small pedigrees and a large number of markers. It is used in the analysis of  genetic linkage . [ 1 ] [ 2 ]


Cover of an unknown issue of  Annals of Human Genetics . Image presumed current as of 2005; Retrieved July 14, 2005.


Original file location =  [1] ; found on page  http://www.blackwellpublishing.com/journal.asp?ref=0003-4800 .


Annals of Human Genetics


Periodical front cover showing original artword, publisher logo and title; cover depicted is the single standard cover for the periodical during at least the period of 2005-07 to 2007-09.


Yes


Depicting the subject of the article  Annals of Human Genetics , the standard cover for this periodical shown to provide visual context and recognition when encountered physically.


No. Potential replacement would be a photograph of an issue of the periodical; however, this periodical is usually found in public and private institutional libraries, which typically have restrictions on the use of photographic equipment on library premises.


Created by me, using elements of US government drawing at  Human Genome Project


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A  birth defect [ a ]  is an abnormal condition that is present at  birth , regardless of its cause. [ 2 ]  Birth defects may result in  disabilities  that may be  physical ,  intellectual , or  developmental . [ 2 ]  The disabilities can range from mild to severe. [ 6 ]  Birth defects are divided into two main types: structural disorders in which problems are seen with the shape of a body part and  functional disorders  in which problems exist with how a body part works. [ 3 ]  Functional disorders include  metabolic  and  degenerative disorders . [ 3 ]  Some birth defects include both structural and functional disorders. [ 3 ]


Birth defects may result from  genetic  or  chromosomal disorders , exposure to certain medications or chemicals, or certain  infections during pregnancy . [ 4 ]  Risk factors include  folate deficiency ,  drinking alcohol  or  smoking  during pregnancy, poorly controlled  diabetes , and a mother over the age of 35 years old. [ 5 ] [ 6 ]  Many are believed to involve multiple factors. [ 6 ]  Birth defects may be visible at birth or diagnosed by  screening tests . [ 10 ]  A number of defects can be detected before birth by different  prenatal tests . [ 10 ]


Treatment varies depending on the defect in question. [ 7 ]  This may include  therapy , medication, surgery, or  assistive technology . [ 7 ]  Birth defects affected about 96 million people as of 2015 [update] . [ 11 ]  In the United States, they occur in about 3% of newborns. [ 8 ]  They resulted in about 628,000 deaths in 2015, down from 751,000 in 1990. [ 9 ] [ 12 ]  The types with the greatest numbers of deaths are  congenital heart disease  (303,000), followed by  neural tube defects  (65,000). [ 9 ]


Much of the language used for describing congenital conditions antedates  genome mapping , and structural conditions are often considered separately from other congenital conditions. Many metabolic conditions are now known to have subtle structural expression, and structural conditions often have genetic links. Still, congenital conditions are often classified on a structural basis, organized when possible by primary organ system affected. [ citation needed ]


Several terms are used to describe congenital abnormalities. (Some of these are also used to describe noncongenital conditions, and more than one term may apply in an individual condition.)


A limb anomaly is called a  dysmelia . These include all forms of limbs anomalies, such as  amelia ,  ectrodactyly ,  phocomelia ,  polymelia ,  polydactyly ,  syndactyly ,  polysyndactyly ,  oligodactyly ,  brachydactyly ,  achondroplasia , congenital  aplasia  or  hypoplasia ,  amniotic band syndrome , and  cleidocranial dysostosis . [ 17 ]


Congenital heart defects  include  patent ductus arteriosus ,  atrial septal defect ,  ventricular septal defect , and  tetralogy of Fallot .


Congenital anomalies of the nervous system include neural tube defects such as  spina bifida ,  encephalocele , and  anencephaly . Other congenital anomalies of the nervous system include the  Arnold–Chiari malformation , the  Dandy–Walker malformation ,  hydrocephalus ,  microencephaly ,  megalencephaly ,  lissencephaly ,  polymicrogyria ,  holoprosencephaly , and  agenesis of the corpus callosum . [ 18 ]


Congenital anomalies of the  gastrointestinal system  include numerous forms of  stenosis  and  atresia , and perforation, such as  gastroschisis . [ 19 ]


Congenital anomalies of the kidney and urinary tract include renal parenchyma, kidneys, and urinary collecting system. [ 20 ]


Defects can be bilateral or unilateral, and different defects often coexist in an individual child. [ 21 ]


A  congenital metabolic disease  is also referred to as an  inborn error of metabolism . Most of these are  single-gene defects , usually heritable. Many affect the structure of body parts, but some simply affect the function. [ 22 ]


Other well-defined genetic conditions may affect the production of hormones, receptors, structural proteins, and ion channels. [ citation needed ]


The mother's consumption of alcohol during pregnancy can cause a continuum of various permanent birth defects: craniofacial abnormalities, [ 23 ]  brain damage, [ 24 ]  intellectual disability, [ 25 ]  heart disease, kidney abnormality, skeletal anomalies, ocular abnormalities. [ 26 ]


The prevalence of children affected is estimated at least 1% in U.S. [ 27 ]  as well in Canada.


Very few studies have investigated the links between paternal alcohol use and offspring health. [ 28 ]


However, recent animal research has shown a correlation between paternal alcohol exposure and decreased offspring birth weight. Behavioral and cognitive disorders, including difficulties with learning and memory, hyperactivity, and lowered stress tolerance have been linked to paternal alcohol ingestion. [ 29 ]  The compromised stress management skills of animals whose male parent was exposed to alcohol are similar to the exaggerated responses to stress that children with  fetal alcohol syndrome  display because of maternal alcohol use. These birth defects and behavioral disorders were found in cases of both long- and short-term paternal alcohol ingestion. [ 30 ] [ 31 ]  In the same animal study, paternal alcohol exposure was correlated with a significant difference in organ size and the increased risk of the offspring displaying  ventricular septal defects  at birth. [ 31 ]


Substances whose  toxicity  can cause congenital disorders are called  teratogens , and include certain pharmaceutical and recreational  drugs in pregnancy , as well as many  environmental toxins in pregnancy . [ 32 ]


A review published in 2010 identified six main teratogenic mechanisms associated with medication use:  folate antagonism ,  neural crest cell  disruption,  endocrine disruption ,  oxidative stress ,  vascular  disruption, and specific receptor- or enzyme-mediated teratogenesis. [ 33 ]


An estimated 10% of all birth defects are caused by prenatal exposure to a teratogenic agent. [ 34 ]  These exposures include medication or drug exposures, maternal infections and diseases, and environmental and occupational exposures. Paternal smoking has also been linked to an increased risk of birth defects and childhood cancer for the offspring, where the paternal germline undergoes oxidative damage due to cigarette use. [ 35 ] [ 36 ]  Teratogen-caused birth defects are potentially preventable. Nearly 50% of pregnant women have been exposed to at least one medication during gestation. [ 37 ]  During pregnancy, a woman can also be exposed to teratogens from contaminated clothing or toxins within the seminal fluid of a partner. [ 38 ] [ 30 ] [ 39 ]  An additional study found that of 200 individuals referred for genetic counseling for a teratogenic exposure, 52% were exposed to more than one potential teratogen. [ 40 ]


The  United States Environmental Protection Agency  studied 1,065 chemical and drug substances in their ToxCast program (part of the  CompTox Chemicals Dashboard ) using  in silico  modeling and a human  pluripotent   stem cell -based assay to predict  in vivo  developmental intoxicants based on changes in cellular  metabolism  following chemical exposure. Findings of the study published in 2020 were that 19% of the 1065 chemicals yielded a prediction of  developmental toxicity . [ 41 ]


Probably, the most well-known teratogenic drug is  thalidomide . It was developed near the end of the 1950s by Chemie Grünenthal as a  sleep-inducing aid  and  antiemetic . Because of its ability to prevent nausea, it was prescribed for pregnant women in almost 50 countries worldwide between 1956 and 1962. [ 42 ]  Until  William McBride  published the study leading to its withdrawal from the market in 1961, about 8,000 to 10,000 severely malformed children were born. The most typical disorders induced by thalidomide were reductional deformities of the long bones of the extremities.  Phocomelia , otherwise a rare deformity, therefore helped to recognise the teratogenic effect of the new drug. Among other malformations caused by thalidomide were those of ears, eyes, brain, kidney, heart, and digestive and respiratory tracts; 40% of the prenatally affected children died soon after birth. [ 42 ]  As thalidomide is used today as a treatment for  multiple myeloma  and  leprosy , several births of affected children were described in spite of the strictly required use of contraception among female patients treated by it. [ citation needed ]


Vitamin A  is the sole vitamin that is embryotoxic even in a therapeutic dose, for example in  multivitamins , because its metabolite,  retinoic acid , plays an important role as a signal molecule in the development of several tissues and organs. Its natural precursor,  β-carotene , is considered safe, whereas the consumption of animal liver can lead to malformation, as the liver stores lipophilic vitamins, including retinol. [ 42 ]   Isotretinoin  (13-cis-retinoic-acid; brand name Roaccutane), vitamin A analog, which is often used to treat severe  acne , is such a strong teratogen that just a single dose taken by a pregnant woman (even  transdermally ) may result in serious birth defects. Because of this effect, most countries have systems in place to ensure that it is not given to pregnant women and that the patient is aware of how important it is to prevent pregnancy during and at least one month after treatment. Medical guidelines also suggest that pregnant women should limit vitamin A intake to about 700  μg /day, as it has teratogenic potential when consumed in excess. [ 43 ] [ 44 ]  Vitamin A and similar substances can induce spontaneous abortions, premature births, defects of eyes ( microphthalmia ), ears, thymus, face deformities, and neurological ( hydrocephalus ,  microcephalia ) and cardiovascular defects, as well as  intellectual disability . [ 42 ]


Tetracycline , an  antibiotic , should never be prescribed to women of reproductive age or to children, because of its negative impact on  bone  mineralization and  teeth mineralization . The "tetracycline teeth" have brown or grey colour as a result of a defective development of both the  dentine  and the  enamel of teeth . [ 42 ]


Several  anticonvulsants  are known to be highly teratogenic.  Phenytoin , also known as diphenylhydantoin, along with  carbamazepine , is responsible for the  fetal hydantoin syndrome , which may typically include broad nose base, cleft lip and/or palate,  microcephalia , nails and fingers  hypoplasia ,  intrauterine growth restriction , and intellectual disability.  Trimethadione  taken during pregnancy is responsible for the  fetal trimethadione syndrome , characterized by craniofacial, cardiovascular, renal, and spine malformations, along with a delay in mental and physical development.  Valproate  has  antifolate  effects, leading to  neural tube  closure-related defects such as spina bifida. Lower  IQ  and  autism  have recently also been reported as a result of intrauterine valproate exposure. [ 42 ]


Hormonal contraception  is considered harmless for the embryo. Peterka and Novotná [ 42 ]  do, however, state that synthetic  progestins  used to prevent miscarriage in the past frequently caused masculinization of the outer reproductive organs of female newborns due to their  androgenic  activity.  Diethylstilbestrol  is a synthetic  estrogen  used from the 1940s to 1971, when the prenatal exposition has been linked to the  clear-cell adenocarcinoma of the vagina . Following studies showed elevated risks for other tumors and congenital malformations of the sex organs for both sexes. [ citation needed ]


All  cytostatics  are strong teratogens;  abortion  is usually recommended when pregnancy is discovered during or before chemotherapy.  Aminopterin , a cytostatic drug with anti folate  effect, was used during the 1950s and 1960s to induce  therapeutic abortions . In some cases, the abortion did not happen, but the newborns had a fetal aminopterin syndrome consisting of growth retardation,  craniosynostosis , hydrocephalus, facial dismorphities, intellectual disability, or leg deformities [ 42 ] [ 45 ]


Drinking water  is often a medium through which harmful toxins travel. Heavy metals, elements, nitrates, nitrites, and fluoride can be carried through water and cause congenital disorders. [ 46 ]


Nitrate, which is found mostly in drinking water from ground sources, is a powerful teratogen. A case-control study in rural Australia that was conducted following frequent reports of prenatal mortality and congenital malformations found that those who drank the nitrate-containing groundwater, as opposed to rain water, ran the risk of giving birth to children with central nervous system disorders, muscoskeletal defects, and cardiac defects. [ 47 ]


Chlorinated and aromatic solvents such as benzene and trichloroethylene sometimes enter the water supply due to oversights in waste disposal. A case-control study on the area found that by 1986, leukemia was occurring in the children of Woburn, Massachusetts, at a rate that was four times the expected rate of incidence. Further investigation revealed a connection between the high occurrence of leukemia and an error in water distribution that delivered water to the town with significant contamination with manufacturing waste containing trichloroethylene. [ 48 ]
As an  endocrine disruptor ,  DDT  was shown to induce  miscarriages , interfere with the development of the  female reproductive system , cause the  congenital hypothyroidism , and suspectably  childhood obesity . [ 42 ]


Fluoride, when transmitted through water at high levels, can also act as a teratogen. Two reports on fluoride exposure from China, which were controlled to account for the education level of parents, found that children born to parents who were exposed to 4.12 ppm fluoride grew to have IQs that were, on average, seven points lower than their counterparts whose parents consumed water that contained 0.91 ppm fluoride. In studies conducted on rats, higher fluoride in drinking water led to increased acetylcholinesterase levels, which can alter prenatal brain development. The most significant effects were noted at a level of 5 ppm. [ 49 ]


The fetus is even more susceptible to damage from carbon monoxide intake, which can be harmful when inhaled during pregnancy, usually through first- or second-hand tobacco smoke. The concentration of carbon monoxide in the infant born to a nonsmoking mother is around 2%, and this concentration drastically increases to a range of 6%–9% if the mother smoked tobacco. Other possible sources of prenatal carbon monoxide intoxication are exhaust gas from combustion motors, use of dichloromethane (paint thinner, varnish removers) in enclosed areas, defective gas water heaters, indoor barbeques, open flames in poorly ventilated areas, and atmospheric exposure in highly polluted areas. [ 50 ]  Exposure to carbon monoxide at toxic levels during the first two trimesters of pregnancy can lead to intrauterine growth restriction, leading to a baby who has stunted growth and is born smaller than 90% of other babies at the same gestational age. The effect of chronic exposure to carbon monoxide can depend on the stage of pregnancy in which the mother is exposed. Exposure during the embryonic stage can have neurological consequences, such as telencephalic dysgenesis, behavioral difficulties during infancy, and reduction of cerebellum volume. Also, possible skeletal defects could result from exposure to carbon monoxide during the embryonic stage, such as hand and foot malformations,  hip dysplasia , hip subluxation, agenesis of a limb, and inferior maxillary atresia with  glossoptosis . Also, carbon monoxide exposure between days 35 and 40 of embryonic development can lead to an increased risk of the child developing a cleft palate. Exposure to carbon monoxide or polluted ozone exposure can also lead to cardiac defects of the ventrical septal, pulmonary artery, and heart valves. [ 51 ]  The effects of carbon monoxide exposure are decreased later in fetal development during the fetal stage, but they may still lead to  anoxic   encephalopathy . [ 52 ]


Industrial pollution can also lead to congenital defects. [ 53 ]  Over a period of 37 years, the  Chisso  Corporation, a petrochemical and plastics company, contaminated the waters of  Minamata Bay  with an estimated 27 tons of  methylmercury , contaminating the local water supply. This led many people in the area to develop what became known as the " Minamata disease ". Because methylmercury is a teratogen, the  mercury poisoning  of those residing by the bay resulted in neurological defects in the offspring. Infants exposed to mercury poisoning  in utero  showed predispositions to  cerebral palsy ,  ataxia , inhibited psychomotor development, and intellectual disability. [ 54 ]


Landfill sites have been shown to have adverse effects on fetal development. Extensive research has shown that landfills have several negative effects on babies born to mothers living near landfill sites: low birth weight, birth defects, spontaneous abortion, and fetal and infant mortality. Studies done around the  Love Canal  site near Niagara Falls and the  Lipari Landfill  in New Jersey have shown a higher proportion of low birth-weight babies than communities farther away from landfills. A study done in California showed a positive correlation between time and quantity of dumping and low birth weights and neonatal deaths. A study in the United Kingdom showed a correlation between pregnant women living near landfill sites and an increased risk of congenital disorders, such as neural tube defects,  hypospadias ,  epispadia , and  abdominal wall defects , such as  gastroschisis  and exomphalos. A study conducted on a Welsh community also showed an increased incidence of gastroschisis. Another study on 21 European hazardous-waste sites showed that those living within 3 km had an increased risk of giving birth to infants with birth defects and that as distance from the land increased, the risk decreased. These birth defects included neural tube defects, malformations of the cardiac septa, anomalies of arteries and veins, and chromosomal anomalies. [ 55 ]  Looking at communities that live near landfill sites brings up environmental justice. A vast majority of sites are located near poor, mostly black, communities. For example, between the early 1920s and 1978, about 25% of Houston's population was black. However, over 80% of landfills and incinerators during this time were located in these black communities. [ 56 ]


Another issue regarding  environmental justice  is  lead poisoning . A fetus exposed to lead during the pregnancy can result in learning difficulties and slowed growth. Some paints (before 1978) and pipes contain lead. Therefore, pregnant women who live in homes with lead paint inhale the dust containing lead, leading to lead exposure in the fetus. When lead pipes are used for drinking water and cooking water, this water is ingested, along with the lead, exposing the fetus to this toxin. This issue is more prevalent in poorer communities because more well-off families are able to afford to have their homes repainted and pipes renovated. [ 57 ]


Endometriosis can impact a woman's  fetus , causing a 30% higher risk for congenital malformations and a 50% higher risk of  neonates  being under-sized for their gestational age. [ 58 ]


Paternal smoking prior to conception has been linked with the increased risk of congenital abnormalities in offspring. [ 28 ]


Smoking causes DNA mutations in the germline of the father, which can be inherited by the offspring. Cigarette smoke acts as a chemical mutagen on germ cell DNA. The germ cells suffer oxidative damage, and the effects can be seen in altered mRNA production, infertility issues, and side effects in the embryonic and fetal stages of development. This  oxidative damage  may result in epigenetic or genetic modifications of the father's germline. Fetal  lymphocytes  have been damaged as a result of a father's smoking habits prior to conception. [ 36 ] [ 38 ]


Correlations between paternal smoking and the increased risk of offspring developing childhood cancers (including acute  leukemia ,  brain tumors , and  lymphoma ) before age five have been established. Little is currently known about how paternal smoking damages the fetus, and what window of time in which the father smokes is most harmful to offspring. [ 36 ]


A  vertically transmitted infection  is an  infection  caused by  bacteria ,  viruses , or in rare cases,  parasites   transmitted  directly from the mother to an  embryo ,  fetus , or baby during pregnancy or childbirth. [ 59 ]


Congenital disorders were initially believed to be the result of only hereditary factors. However, in the early 1940s, Australian pediatric ophthalmologist  Norman Gregg  began recognizing a pattern in which the infants arriving at his surgery were developing congenital cataracts at a higher rate than those who developed it from hereditary factors. [ 60 ]  On October 15, 1941, Gregg delivered a paper that explained his findings-68 out of the 78 children with congenital cataracts had been exposed  in utero  to rubella due to an outbreak in Australian army camps. These findings confirmed, to Gregg, that, in fact, environmental causes for congenital disorders could exist. [ citation needed ]


Rubella  is known to cause abnormalities of the eye, internal ear, heart, and sometimes the teeth. More specifically, fetal exposure to rubella during weeks five to ten of development (the sixth week particularly) can cause  cataracts  and  microphthalmia  in the eyes. If the mother is infected with rubella during the ninth week, a crucial week for internal ear development, destruction of the  organ of Corti  can occur, causing deafness. In the heart, the  ductus arteriosus  can remain after birth, leading to hypertension. Rubella can also lead to atrial and ventricular septal defects in the heart. If exposed to rubella in the second trimester, the fetus can develop central nervous system malformations. However, because infections of rubella may remain undetected, misdiagnosed, or unrecognized in the mother, and/or some abnormalities are not evident until later in the child's life, precise incidence of birth defects due to rubella are not entirely known. The timing of the mother's infection during fetal development determines the risk and type of birth defect. As the embryo develops, the risk of abnormalities decreases. If exposed to the rubella virus during the first four weeks, the risk of malformations is 47%. Exposure during weeks five through eight creates a 22% chance, while weeks 9–12, a 7% chance exists, followed by 6% if the exposure is during the 13th-16th weeks. Exposure during the first eight weeks of development can also lead to premature birth and fetal death. These numbers are calculated from immediate inspection of the infant after birth. Therefore, mental defects are not accounted for in the percentages because they are not evident until later in the child's life. If they were to be included, these numbers would be much higher. [ 61 ]


Other infectious agents include  cytomegalovirus , the  herpes simplex virus ,  hyperthermia ,  toxoplasmosis , and  syphilis . Maternal exposure to cytomegalovirus can cause  microcephaly , cerebral calcifications, blindness,  chorioretinitis  (which can cause blindness),  hepatosplenomegaly , and meningoencephalitis in fetuses. [ 61 ]  Microcephaly is a disorder in which the fetus has an atypically small head, [ 62 ]  cerebral calcifications means certain areas of the brain have atypical calcium deposits, [ 63 ]  and meningoencephalitis is the enlargement of the brain. All three disorders cause abnormal brain function or intellectual disability. Hepatosplenomegaly is the enlargement of the liver and spleen which causes digestive problems. [ 64 ]  It can also cause some  kernicterus  and  petechiae . Kernicterus causes yellow pigmentation of the skin, brain damage, and deafness. [ 65 ]  Petechaie is when the capillaries bleed resulting in red/purple spots on the skin. [ 66 ]  However, cytomegalovirus is often fatal in the embryo. The  Zika virus  can also be transmitted from the pregnant mother to her baby and cause microcephaly. [ citation needed ]


The herpes simplex virus can cause  microcephaly , microphthalmus (abnormally small eyeballs), [ 67 ]  retinal dysplasia,  hepatosplenomegaly , and intellectual disability. [ 61 ]  Both microphthalmus and retinal dysplasia can cause blindness. However, the most common symptom in infants is an inflammatory response that develops during the first three weeks of life. [ 61 ]  Hyperthermia causes  anencephaly , which is when part of the brain and skull are absent in the infant. [ 61 ] [ 68 ]  Mother exposure to toxoplasmosis can cause cerebral calcification, hydrocephalus (causes mental disabilities), [ 69 ]  and intellectual disability in infants. Other birth abnormalities have been reported as well, such as chorioretinitis, microphthalmus, and ocular defects. Syphilis causes congenital deafness, intellectual disability, and diffuse fibrosis in organs, such as the liver and lungs, if the embryo is exposed. [ 61 ]


For example, a lack of  folic acid , a B vitamin, in the diet of a mother can cause cellular  neural tube  deformities that result in spina bifida. Congenital disorders such as a neural tube deformity can be prevented by 72% if the mother consumes 4 mg of folic acid before the conception and after twelve weeks of pregnancy. [ 70 ]  Folic acid, or vitamin B 9 , aids the development of the foetal nervous system. [ 70 ]


Studies with mice have found that food deprivation of the male mouse prior to conception leads to the offspring displaying significantly lower blood glucose levels. [ 71 ]


External physical shocks or constraints due to growth in a restricted space may result in unintended deformation or separation of cellular structures resulting in an abnormal final shape or damaged structures unable to function as expected. An example is  Potter syndrome  due to  oligohydramnios . This finding is important for future understanding of how genetics may predispose individuals for diseases such as obesity, diabetes, and cancer. [ 72 ]


For multicellular organisms that develop in a  womb , the physical interference or presence of other similarly developing organisms such as  twins  can result in the two cellular masses being integrated into a larger whole, with the combined cells attempting to continue to develop in a manner that satisfies the intended growth patterns of both cell masses. [ 73 ]  The two cellular masses can compete with each other, and may either duplicate or merge various structures. This results in conditions such as  conjoined twins , and the resulting merged organism may die at birth when it must leave the life-sustaining environment of the womb and must attempt to sustain its biological processes independently. [ citation needed ]


Genetic causes of birth defects include  inheritance  of abnormal  genes  from the mother or the father, as well as new  mutations  in one of the  germ cells  that gave rise to the fetus. Male germ cells mutate at a much faster rate than female germ cells, and as the father ages, the DNA of the germ cells mutates quickly. [ 35 ] [ 74 ]  If an egg is fertilized with sperm that has damaged DNA, a possibility exists that the fetus could develop abnormally. [ 74 ] [ 75 ]


Genetic disorders are all congenital (present at birth), though they may not be expressed or recognized until later in life. Genetic disorders may be grouped into single-gene defects, multiple-gene disorders, or  chromosomal defects . Single-gene defects may arise from abnormalities of both copies of an  autosomal  gene (a  recessive  disorder) or of only one of the two copies (a  dominant  disorder). Some conditions result from deletions or abnormalities of a few genes located contiguously on a chromosome. Chromosomal disorders involve the loss or duplication of larger portions of a chromosome (or an entire chromosome) containing hundreds of genes. Large chromosomal abnormalities always produce effects on many different body parts and organ systems. [ citation needed ]


Non-genetic defects in sperm cells, such as deformed  centrioles  and other components in the tail and neck of the sperm which are important for the embryonic development, may result in defects. [ 76 ] [ 77 ]


A low  socioeconomic status  in a deprived neighborhood may include exposure to "environmental stressors and risk factors". [ 78 ]  Socioeconomic inequalities are commonly measured by the Cartairs-Morris score, Index of Multiple Deprivation, Townsend deprivation index, and the Jarman score. [ 79 ]  The Jarman score, for example, considers "unemployment, overcrowding, single parents, under-fives, elderly living alone, ethnicity, low social class and residential mobility". [ 79 ]  In Vos' meta-analysis these indices are used to view the effect of low SES neighborhoods on maternal health. In the meta-analysis, data from individual studies were collected from 1985 up until 2008. [ 79 ]  Vos concludes that a correlation exists between prenatal adversities and deprived neighborhoods. [ 79 ]  Other studies have shown that low SES is closely associated with the development of the fetus in utero and growth retardation. [ 80 ]  Studies also suggest that children born in low SES families are "likely to be born prematurely, at low birth weight, or with asphyxia, a birth defect, a disability, fetal alcohol syndrome, or AIDS". [ 80 ]  Bradley and Corwyn also suggest that congenital disorders arise from the mother's lack of nutrition, a poor lifestyle, maternal substance abuse and "living in a neighborhood that contains hazards affecting fetal development (toxic waste dumps)". [ 80 ]  In a meta-analysis that viewed how inequalities influenced maternal health, it was suggested that deprived neighborhoods often promoted behaviors such as smoking, drug and alcohol use. [ 78 ]  After controlling for socioeconomic factors and ethnicity, several individual studies demonstrated an association with outcomes such as perinatal mortality and preterm birth. [ 78 ]


For the survivors of the  atomic bombing of Hiroshima  and  Nagasaki , who are known as the  Hibakusha , no statistically demonstrable increase of birth defects/congenital malformations was found among their later conceived children, or found in the later conceived children of cancer survivors who had previously received  radiotherapy . [ 81 ] [ 82 ] [ 83 ] [ 84 ]
The surviving women of Hiroshima and Nagasaki who were able to conceive, though exposed to substantial amounts of radiation, later had children with no higher incidence of abnormalities/birth defects than in the Japanese population as a whole. [ 85 ] [ 86 ]


Relatively few studies have researched the effects of paternal radiation exposure on offspring. Following the  Chernobyl  disaster, it was assumed in the 1990s that the germ line of irradiated fathers suffered  minisatellite  mutations in the DNA, which was inherited by descendants. [ 30 ] [ 87 ]  More recently, however, the World Health Organization states, "children conceived before or after their father's exposure showed no statistically significant differences in mutation frequencies". [ 88 ]  This  statistically insignificant  increase was also seen by independent researchers analyzing the children of the  liquidators . [ 89 ]  Animal studies have shown that incomparably  massive  doses of X-ray irradiation of male mice resulted in birth defects of the offspring. [ 38 ]


In the 1980s, a relatively high prevalence of pediatric leukemia cases in children living near a nuclear processing plant in West Cumbria, UK, led researchers to investigate whether the cancer was a result of paternal radiation exposure. A significant association between paternal irradiation and offspring cancer was found, but further research areas close to other nuclear processing plants did not produce the same results. [ 38 ] [ 30 ]  Later this was determined to be the  Seascale cluster  in which the leading hypothesis is the influx of foreign workers, who have a different rate of leukemia within their race than the British average, resulted in the observed cluster of 6 children more than expected around Cumbria. [ 90 ]


Certain birth complications can occur more often in  advanced maternal age  (greater than 35 years). Complications include fetal growth restriction, preeclampsia, placental abruption, pre-mature births, and stillbirth. These complications not only may put the child at risk, but also the mother. [ 91 ]


The effects of the father's age on offspring are not yet well understood and are studied far less extensively than the effects of the mother's age. [ 92 ]  Fathers contribute proportionally more DNA mutations to their offspring via their germ cells than the mother, with the paternal age governing how many mutations are passed on. This is because, as humans age, male germ cells acquire mutations at a much faster rate than female germ cells. [ 35 ] [ 38 ] [ 74 ]


Around a 5% increase in the incidence of  ventricular septal defects , atrial septal defects, and  patent ductus arteriosus  in offspring has been found to be correlated with advanced paternal age. Advanced paternal age has also been linked to increased risk of  achondroplasia  and  Apert syndrome . Offspring born to fathers under the age of 20 show increased risk of being affected by patent ductus arteriosus, ventricular septal defects, and the  tetralogy of Fallot . It is hypothesized that this may be due to environmental exposures or lifestyle choices. [ 92 ]


Research has found that there is a correlation between advanced paternal age and risk of birth defects such as  limb anomalies , syndromes involving multiple systems, and  Down syndrome . [ 74 ] [ 35 ] [ 93 ]  Recent studies have concluded that 5-9% of Down syndrome cases are due to paternal effects, but these findings are controversial. [ 74 ] [ 75 ] [ 35 ] [ 94 ]


There is concrete evidence that advanced paternal age is associated with the increased likelihood that a mother will have a  miscarriage  or that  fetal death  will occur. [ 74 ]


Although significant progress has been made in identifying the etiology of some birth defects, approximately 65% have no known or identifiable cause. [ 34 ]  These are referred to as sporadic, a term that implies an unknown cause, random occurrence regardless of maternal living conditions, [ 95 ]  and a low recurrence risk for future children. For 20-25% of anomalies there seems to be a "multifactorial" cause, meaning a complex interaction of multiple minor genetic anomalies with environmental risk factors. Another 10–13% of anomalies have a purely environmental cause (e.g. infections, illness, or drug abuse in the mother). Only 12–25% of anomalies have a purely genetic cause. Of these, the majority are  chromosomal anomalies . [ 96 ]


Congenital disorders are not limited to humans and can be found in a variety of other species, including cattle. One such condition is called schistosomus reflexus and is defined by spinal inversion, exposure of abdominal viscera, and limb abnormalities. [ 97 ]


Folate supplements decrease the risk of neural tube defects. Tentative evidence supports the role of  L-arginine  in decreasing the risk of  intrauterine growth restriction . [ 98 ]


Newborn screening tests  were introduced in the early 1960s and initially dealt with just two disorders. Since then  tandem mass spectrometry ,  gas chromatography–mass spectrometry , and DNA analysis has made it possible for a much larger range of disorders to be screened. Newborn screening mostly measures metabolite and enzyme activity using a dried blood spot sample. [ 99 ]  Screening tests are carried out in order to detect serious disorders that may be treatable to some extent. [ 100 ]  Early diagnosis makes possible the readiness of therapeutic dietary information, enzyme replacement therapy and organ transplants. [ 101 ]  Different countries support the screening for a number of metabolic disorders ( inborn errors of metabolism  (IEM)), and genetic disorders including  cystic fibrosis  and  Duchenne muscular dystrophy . [ 100 ] [ 102 ]
Tandem mass spectroscopy can also be used for IEM, and investigation of sudden infant death, and shaken baby syndrome. [ 100 ]


Screening can also be carried out  prenatally  and can include  obstetric ultrasonography  to give scans such as the  nuchal scan .  3D ultrasound  scans can give detailed information of structural anomalies. [ citation needed ]


Congenital anomalies resulted in about 632,000 deaths per year in 2013 down from 751,000 in 1990. [ 12 ]  The types with the greatest death are  congenital heart defects  (323,000), followed by  neural tube defects  (69,000). [ 12 ]


Many studies have found that the frequency of occurrence of certain congenital malformations depends on the sex of the child (table). [ 104 ] [ 105 ] [ 106 ] [ 107 ] [ 108 ]  For example, pyloric stenosis occurs more often in males while congenital hip dislocation is four to five times more likely to occur in females. Among children with one kidney, there are approximately twice as many males, whereas among children with three kidneys there are approximately 2.5 times more females. The same pattern is observed among infants with excessive number of ribs, vertebrae, teeth and other organs which in a process of evolution have undergone reduction—among them there are more females. Contrarily, among the infants with their scarcity, there are more males. Anencephaly is shown to occur approximately twice as frequently in females. [ 109 ]  The number of boys born with 6 fingers is two times higher than the number of girls. [ 110 ]  Now various techniques are available to detect congenital anomalies in fetus before birth. [ 111 ]


About 3% of newborns have a "major physical anomaly", meaning a physical anomaly that has cosmetic or functional significance. [ 112 ]
Physical congenital abnormalities are the leading cause of infant mortality in the United States, accounting for more than 20% of all infant deaths. Seven to ten percent of all children [ clarification needed ]  will require extensive medical care to diagnose or treat a birth defect. [ 113 ]


P. M. Rajewski and A. L. Sherman (1976) have analyzed the frequency of congenital anomalies in relation to the system of the organism. Prevalence of men was recorded for the anomalies of phylogenetically younger organs and systems. [ 114 ]


In respect of an etiology, sexual distinctions can be divided on appearing before and after differentiation of male's gonads during embryonic development, which begins from the eighteenth week. The testosterone level in male embryos thus raises considerably. [ 118 ]  The subsequent hormonal and physiological distinctions of male and female embryos can explain some sexual differences in frequency of congenital defects. [ 119 ]  It is difficult to explain the observed differences in the frequency of birth defects between the sexes by the details of the reproductive functions or the influence of environmental and social factors. [ citation needed ]


The CDC and National Birth Defect Project studied the incidence of birth defects in the US. Key findings include: [ citation needed ]


List of  congenital disorders


1q21.1  copy number variations  (CNVs) [ 1 ]  are rare aberrations of human  chromosome 1 .


In a common situation a human cell has one pair of identical chromosomes on chromosome 1. With the 1q21.1 CNVs one chromosome of the pair is not complete because a part of the sequence of the chromosome is missing, or overcomplete, because some parts of the sequence are duplicated. The result is that one chromosome is of normal length and the other one is too long or too short. [ citation needed ]


The structure of 1q21.1 is complex. The area has a size of approximately 6 Mega base  (Mb) (from 141.5 Mb to 147.9 Mb). Within 1q21.1 there are two areas where the CNVs can be found: the proximal area or TAR area (144.1 to 144.5) and the distal area (144.7 to 145.9). A 1q21.1 CNV will commonly be found in one of these areas, but an overlap with the other area or parts that are outside these areas are possible. 1q21.1 has multiple repetitions of the same structure: Only 25% of the structure is not duplicated. There are several gaps in the sequence. There is no further information available about the DNA-sequence in those areas up till now. The gaps represent approximately 700 Kilobase. New genes are expected in the gaps. The area of 1q21.1 is one of the most difficult parts of the human genome to map. [ citation needed ]


CNVs occur due to  non-allelic homologous recombination  mediated by  low copy repeats  (sequentially similar regions), commonly found in 1q21.1. [ citation needed ]


Four separate forms of 1q21.1 CNVs are mentioned in literature. [ citation needed ]


The CNVs lead to a very variable  phenotype  and the manifestations in individuals are quite variable. Some people who have a CNV can function in a normal way, while others have symptoms of mental retardation and various physical anomalies. [ citation needed ]


Meiosis  is the process of dividing cells in humans. In meiosis, the chromosome pairs splits and a representative of each pair goes to one daughter cell. In this way the number of chromosomes will be halved in each cell, while all the parts on the chromosome (genes) remain, after being randomized. Which information of the parent cell ends up in the daughter cell is purely decided by chance. Besides this random process, there is a second random process. In this second random process the DNA will be scrambled in a way that pieces are omitted ( deletion ), added ( duplication ), moved from one place to another ( translocation ) and inverted ( inversion ). This is a common process, which leads to about 0,4% variation in the DNA. It explains why even identical twins are not genetically 100% identical. [ citation needed ]


Problem of the second random process is that genetic mistakes can occur. Especially due to the deletion and duplication process, the chromosomes that come together in a new cell may be shorter or longer. The result of this spontaneous change in the structure of DNA is a so-called  copy number variation . Due to the CNV chromosomes of different sizes can be combined in a new cell. If this occurs around conception, the result will be a first cell of a human with a genetic variation. This can be either positive or negative. In positive cases this new human will be capable of a special skill that is assessed positively, for example, in sports or science. In negative cases, you have to deal with a syndrome or a severe disability, as in this case the 1q21.1 CNVs. [ citation needed ]


Based on the meiotic process, the syndrome may occur in two ways.


Due to this genetic misprint the embryo may experience problems in the development during the first months of pregnancy. Approximately 20 to 40 days after fertilization, something goes wrong in the construction of the body parts and brain, which leads to a chain reaction.


Because of the repetitions in 1q21.1, there is a larger chance on an unequal crossing-over during  meiosis .


Genes related to the proximal area are  HFE2 ,  TXNIP ,  POLR3GL ,  LIX1L ,  RBM8A ,  PEX11B ,  ITGA10 ,  ANKRD35 ,  PIAS3 ,  NUDT17 ,  POLR3C ,  RNF115 ,  CD160 ,  PDZK1 , and  GPR89A
Genes related to the distal area are  PDE4DIP ,  HYDIN2 ,  PRKAB2 ,  PDIA3P ,  FMO5 ,  CHD1L ,  BCL9 ,  ACP6 ,  GJA5 ,  GJA8 ,  NBPF10 ,  GPR89B ,  GPR89C ,  PDZK1P1  and  NBPF11 .


1q21.1 deletion syndrome  is a rare aberration of  chromosome 1 . A human cell has one pair of identical chromosomes on  chromosome 1 . With the 1q21.1 deletion syndrome, one chromosome of the pair is not complete, because a part of the sequence of the chromosome is missing. One chromosome has the normal length and the other is too short.


In 1q21.1, the '1' stands for chromosome 1, the 'q' stands for the long arm of the chromosome and '21.1' stands for the part of the long arm in which the deletion is situated.


The syndrome is a form of the  1q21.1 copy number variations , and it is a deletion in the distal area of the 1q21.1 part. The  CNV  leads to a very variable  phenotype , and the manifestations in individuals are quite variable. Some people who have the syndrome can function in a normal way, while others have symptoms of intellectual impairment and various physical anomalies. [ 1 ]


1q21.1 microdeletion is a very rare chromosomal condition. Only 46 individuals with this deletion have been reported in medical literature as of August 2011. [ 3 ]


Approximately 75% of all children with a 1q21.1 microdeletion exhibit  delayed development , notably in  motor skills  such as sitting, standing, and walking. [ 1 ]  Individuals may have generalized mild  learning difficulties ;  about 30% of those diagnosed with 1q21.1 deletion syndrome are affected. [ 4 ]


Dysmorphic craniofacial  traits are common, however, they are highly varied and thus difficult to identify.  Microcephaly  has been reported in 39% of those with the 1q21.1 deletion. [ 4 ] [ 2 ]


It is not clear whether the list of symptoms is complete. Very little information is known about the syndrome. The syndrome can have completely different effects on members of the same family. [ 1 ] [ 4 ]


Genitourinary  abnormalities include  vesicoureteral reflux ,  hydronephrosis ,  inguinal hernia ,  cryptorchidism , and  genital malformations . There have been two reported cases of  Mayer-Rokitansky-Kuster-Hauser syndrome  alongside 1q21.1 deletion syndrome. [ 6 ] [ 4 ] [ 1 ]


The majority of affected people have normal  neurologic  physical examinations, however  hypotonia  and  tremors  are quite common.  Seizures  affect roughly 16% of children and typically begin during infancy. [ 1 ] [ 4 ]


Possible psychiatric and behavioral abnormalities include  autism spectrum disorder ,  attention deficit hyperactivity disorder , and  mood disorders . Furthermore, distal 1q21.1 microdeletions have been found in 0.2%-0.6% of those with  schizophrenia . [ 4 ] [ 7 ]


A common deletion is between 1.0-1.9Mb. Mefford states that the standard for a deletion is 1.35Mb. [ 8 ]


1q21.1 deletion is  autosomal dominant , with 18%-50% of deletions developing  de novo  and 50%-82%  inherited  from a parent. [ 4 ] [ 1 ]


The majority of affected individuals are missing a 1.35 million  DNA   base pair sequence , also written as 1.35  megabases  (Mb), in the q21.1 region of  chromosome 1 , though the exact size of the deleted region differs. This deletion affects one of each cell's two copies of  chromosome 1 . [ 1 ]


The symptoms caused by a 1q21.1 microdeletion are most likely due to the loss of many  genes  in this region. Researchers are attempting to determine which missing genes may contribute to 1q21.1 deletions' specific characteristics. Because some people with a 1q21.1 microdeletion have no clear associated traits, it is hypothesized that additional  genetic  and  environmental factors  influence the development of symptoms. [ 1 ]


Due to this genetic misprint, the  embryo  may experience problems in the development during the first months of pregnancy. Approximately 20 to 40 days after  fertilization , something goes wrong in the construction of the body parts and brain, which leads to a chain reaction. [ 9 ]


The recurring distal 1.35-Mb  heterozygous  deletion within the position of 145–146.35 Mb in the reference  genome  confirms the diagnosis of 1q21.1 deletion. The  copy number  of sequences can be determined using  chromosome microarrays  or targeted deletion analysis by  fluorescence in situ hybridization . [ 4 ]


22q11.2 microdeletion  shares several characteristics with 1q21.1 microdeletion including  developmental delays ,  learning disabilities ,  intellectual disability , and  behavioral deviations . Those with recurrent 1q21.1 microdeletion, on the other hand, do not have the distinctive facial traits seen in the  22q11.2  microdeletion syndrome. [ 4 ] [ 5 ]


Several examinations should be performed to determine the level of  disability  and the needs of a person with the 1q21.1 deletion. Because of the vast range of symptoms,  ophthalmologic ,  cardiac ,  developmental ,  neurological , and  psychiatric  evaluations may be helpful in identifying the manifestations of 1q21.1 deletion. Different developmental abnormalities of the  skull  and  genitourinary system  can be identified with  brain imaging  and  renal ultrasounds . Consultations with a  medical geneticist  or  genetic counselor  can aid in determining the cause of 1q21.1 deletion. [ 4 ]


Because of the variability in 1q21.1 deletion, management is dependent on specific symptoms.  Occupational  and  physical therapy , as well as special learning programs, may be used to manage symptoms. [ 4 ]


Nonallelic homologous recombination  (NAHR), mediated by  low copy repeats  (LCRs), is a well-known mechanism of  copy number alterations  in an array of  genomic diseases . Individually, these conditions are uncommon, yet collectively, they impact a significant portion of the population. 1q21.1 deletions are estimated to occur in about 0.015% of the population. However, these  CNVs  are incompletely  penetrant , therefore it is likely that the actual prevalence in the general population is greater than the current predictions. [ 5 ]


Statistical research showed that schizophrenia is more common in combination with 1q21.1 deletion syndrome. On the other side, autism is significantly more common with  1q21.1 duplication syndrome . Further research confirmed that the odds on a relation between schizophrenia and deletions at 1q21.1,  3q29 , 15q13.3, 22q11.21 en Neurexin 1 (NRXN1) and duplications at 16p11.2 are at 7.5% or higher. [ 10 ] [ 11 ]


Common variations in the BCL9 gene, which is in the distal area, confer risk of schizophrenia and may also be associated with bipolar disorder and major depressive disorder. [ 12 ]


Research is done on 10–12 genes on 1q21.1 that produce  DUF1220 -locations. DUF1220 is an unknown  protein , which is active in the  neurons  of the  brain  near the  neocortex . Based on research on  apes  and other  mammals , it is assumed that DUF1220 is related to cognitive development (man: 212 locations; chimpanzee: 37 locations; monkey: 30 locations; mouse: 1 location). It appears that the DUF1220-locations on 1q21.1 are in areas that are related to the size and the development of the brain. The aspect of the size and development of the brain is related to autism ( macrocephaly ) and schizophrenia ( microcephaly ). It has been proposed that a deletion or duplication of a gene that produces DUF1220-areas might cause growth and development disorders in the brain [ 13 ]


Another relation between  macrocephaly  with duplications and  microcephaly  with deletions has been seen in research on the HYDIN Paralog or HYDIN2. This part of 1q21.1 is involved in the development of the brain. It is assumed to be a dosage-sensitive gene. When this gene is not available in the 1q21.1 area, it leads to microcephaly. HYDIN2 is a recent duplication (found only in humans) of the HYDIN gene found on 16q22.2. [ 14 ]
Research on the genes  CHD1L  and  PRKAB2  within  lymphoblast  cells [ 15 ]  lead to the conclusion that anomalies appear with the 1q21.1-deletion syndrome:


GJA5 has been identified as the gene that is responsible for the phenotypes observed with congenital heart diseases on the 1q21.1 location. In case of a duplication of GJA5  tetralogy of Fallot  is more common. In case of a deletion other congenital heart diseases than tetralogy of Fallot are more common. [ 16 ]


Aagenaes syndrome  is a  syndrome  characterised by  congenital   hypoplasia  of  lymph vessels , which causes  lymphedema  of the legs and recurrent  cholestasis  in infancy, and slow progress to  hepatic cirrhosis  and  giant cell   hepatitis  with  fibrosis  of the  portal tracts . [ 1 ] [ 2 ]


The genetic cause is due to point mutation (c.-98G>T) in the 5’-untranslated region of Unc-45 myosin chaperone A ( UNC45A ) [ 3 ]  and it is  autosomal recessively   inherited  and the gene is located to chromosome 15q 1,2 . The mutation leads to a loss of function of the protein, which in turn seem to lead to  mislocalization of the hepatobiliary transport proteins BSEP (bile salt export pump) and MRP2 (multidrug resistance-associated protein 2) [ 4 ] .


A common feature of the condition is a generalised lymphoedema from birth or childhood caused by hypoplasia of the  lymphatic vessels  in origin 1 . Approximately one hundred people with this disease are known. [ 5 ]  The condition is particularly frequent in southern  Norway , where more than half the cases are reported, but it is found in patients in other parts of  Europe  and the  United States . [ 6 ]  It is named after Øystein Aagenæs, a Norwegian paediatrician. [ 7 ]  It is also called cholestasis-lymphedema syndrome (CLS). [ 8 ]