The purpose of this study was to understand the role of infection in the origin of chromosomal anomalies linked to neurodevelopmental disorders. In patients with neurodevelopmental disorders, DNA’s from viruses and bacteria including known teratogens were tested against chromosome anomalies known to cause the disorders. Results support a theory that parental infections disrupt elaborate multi-system gene coordination needed for neurodevelopment. Genes essential for neurons, lymphatic drainage, immunity, circulation, angiogenesis, barriers, structure, and chromatin activity were all found close together in polyfunctional clusters that were deleted in neurodevelopmental disorders. These deletions account for immune, circulatory, and structural deficits that accompany neurologic deficits. In deleted clusters, specific and repetitive human DNA matched infections and passed rigorous artifact tests. In some patients, epigenetic driver mutations were found and may be functionally equivalent to deleting a cluster or changing topologic chromatin interactions because they change access to large chromosome segments. In three families, deleted DNA sequences were associated with intellectual deficits and were not included in any database of genomic variants. These sequences were thousands of bp and unequivocally matched foreign DNAs. Analogous homologies were also found in chromosome anomalies of a recurrent neurodevelopmental disorder. Viral and bacterial DNAs that match repetitive or specific human DNA segments are thus proposed to interfere with highly active break repair during meiosis, and sometimes delete polyfunctional clusters, and disable epigenetic drivers. Mis-repaired gametes produce zygotes containing rare chromosome anomalies which cause neurologic disorders and accompanying non-neurologic signs. Neurodevelopmental disorders may be examples of assault on the human genome by foreign DNA with some infections more likely tolerated because they resemble human DNA segments. Further tests of this model await new technology. Graphical representation of the work is shown in figure 1.

Genome, Epigenetics, Neurodevelopmental disorders, Chromosome anomalies, Retrotransposon, Chromosome rearrangement, Neurologic disease, Birth defects, Development, Infection.

The purpose of this study was to understand the role of infection in the origin of chromosomal anomalies linked to neurodevelopmental disorders. In patients with neurodevelopmental disorders, DNA’s from viruses and bacteria including known teratogens were tested against chromosome anomalies known to cause the disorders. Results support a theory that parental infections disrupt elaborate multi-system gene coordination needed for neurodevelopment. Genes essential for neurons, lymphatic drainage, immunity, circulation, angiogenesis, barriers, structure, and chromatin activity were all found close together in polyfunctional clusters that were deleted in neurodevelopmental disorders. These deletions account for immune, circulatory, and structural deficits that accompany neurologic deficits. In deleted clusters, specific and repetitive human DNA matched infections and passed rigorous artifact tests. In some patients, epigenetic driver mutations were found and may be functionally equivalent to deleting a cluster or changing topologic chromatin interactions because they change access to large chromosome segments. In three families, deleted DNA sequences were associated with intellectual deficits and were not included in any database of genomic variants. These sequences were thousands of bp and unequivocally matched foreign DNAs. Analogous homologies were also found in chromosome anomalies of a recurrent neurodevelopmental disorder. Viral and bacterial DNAs that match repetitive or specific human DNA segments are thus proposed to interfere with highly active break repair during meiosis, and sometimes delete polyfunctional clusters, and disable epigenetic drivers. Mis-repaired gametes produce zygotes containing rare chromosome anomalies which cause neurologic disorders and accompanying non-neurologic signs. Neurodevelopmental disorders may be examples of assault on the human genome by foreign DNA with some infections more likely tolerated because they resemble human DNA segments. Further tests of this model await new technology. Graphical representation of the work is shown in figure 1.

Genome, Epigenetics, Neurodevelopmental disorders, Chromosome anomalies, Retrotransposon, Chromosome rearrangement, Neurologic disease, Birth defects, Development, Infection.

An approach to preventing neurodevelopmental disorders is to gain better understanding of how neurodevelopment is coordinated and then to identify environmental and genomic factors that disrupt it. The development of the nervous system requires tight regulation and coordination of multiple functions essential to protect and nourish neurons. As the nervous system develops, the immune system, the circulatory system, cranial and skeletal systems must be synchronized and coordinated. Neurodevelopmental disorders follow the disruption of this coordination.

A significant advance in genome sequence level resolution of balanced cytogenetic abnormalities greatly improves the ability to document changes in regulation and dosage for genes critical to the function of the neurologic system. Based on DNA sequence analyses, some chromosome rearrangements have been identified as causing individual congenital disorders because they disrupt genes essential for normal development [1-3]. There is poor understanding and no effective treatment for many of these overwhelming abnormalities. Signs and symptoms include autism, microcephaly, macrocephaly, behavioral problems, intellectual disability, tantrums, seizures, respiratory problems, spasticity, heart problems, hearing loss, and hallucinations [1]. Because the abnormalities do not correlate well with the outcome, genetic counseling is difficult and uncertain [3].

Several maternal factors are known to interfere with neurodevelopment including infection, and other lifestyle factors that subsequently alter the epigenome, but the genetic events that lead to most neurodevelopmental disorders are not understood [4]. In congenital neurologic disease, inheritance is usually autosomal dominant and the same chromosomal abnormalities occur in every cell. The present work explains how DNA from infections can cause chromosomal deletions, rearrangements, damaged epigenetic controllers and abnormal chromatin topology. Some disorders may arise when infections replicate within the CNS by taking advantage of immune deficiencies such as those traced back to deficient microRNA production [5]. Disseminated infections may interfere with the highly active DNA break repair process required during meiosis, to generate gametes with mis-repaired DNA. In the zygote, this causes chromosome anomalies. The generation of gametes by meiosis is the most active period of recombination. Double strand breaks initiate meiotic recombination and hundreds of double strand breaks occur [6]. The timing and duration of recombination during meiosis differ markedly between males and females. Meiotic recombination in sperm cells occurs continuously after puberty. Errors in spermatogenesis underlie a prevalent and recurrent gene rearrangement that causes Emanuel syndrome. The phenotype of Emanuel syndrome includes intellectual disability, and dysmorphism [2]. In contrast, recombination in ova occurs in fetal life and then meiosis is arrested until puberty. [7].

The exact DNA sequences of known pathogenic rearrangements in unique, familial and recurrent congenital disorders [1-3] makes it possible to test for a mechanism involving microbial DNA at homologous human sequences. For retroviruses, insertion hotspots can significantly change the 3D structure of the genome and disrupt essential chromatin interactions that appear to be long range in two dimensions [8]. Even rare and unique developmental disorders can be screened for homology to infections in the context of altered chromatin structure affecting the immune system, the circulatory system, the formation of the braincirculation barriers and bone development. This screening may assist counseling, diagnosis, prevention, and early intervention.

The results showed that DNAs in some congenital neurodevelopmental disorders closely matches DNA in multiple infections that extend over long linear stretches of human DNA and often involve repetitive human DNA sequences. Neurodevelopmental disorders are proposed to begin when parental infections cause insertions or interfere with meiosis at both repetitive and unique human DNA sequences. The affected sequences are shown to exist as linear clusters of genes closely spaced in two dimensions. Interference from infection can delete or damage human gene clusters and epigenetic regulators that coordinate neurodevelopment. This genomic interference accounts for immune, circulatory, and structural deficits that accompany neurologic deficits. Congenital neurodevelopmental disorders are thus viewed as resulting from an assault on human DNA by microorganisms and an example of the selection of infecting microorganisms based on their similarity to host DNA. However, a linear 2D model is sometimes inadequate to explain certain results and it is important to remember that effects in two dimensions may alter 3D topology as well [8].

Testing and verifying predictions from a viable model may however spur the development of methods for identifying contributions from infections in intractable rare disorders that are not now available. Convergent arguments from testing predictions based on any proposed model might lessen effects of limitations in currently available technology.

DNA sequences from acquired congenital disorders were from published whole genome sequences at chromosome breakpoints and rearrangement sites [1-3]. Comparison to multiple databases of microbial sequences determined whether there were regions of the human genome near recurrent breakage sites that had significant homology. In many cases there was robust evidence that a particular human chromosome rearrangement was pathologic for the congenital disorder and this data was a point of focus.

Hundreds of different private rearrangements in 62 patients with different acquired congenital disorders were tested for homology [9] against non-human sequences from microorganisms known to infect humans as follows: Viruses (taxid: 10239), and retroviruses including HIV- 1 (taxid:11676), and human endogenous retroviruses (Taxids:45617, 87786, 11745, 135201, 166122, 228277and 35268), bacteria (taxid:2), Mycobacteria (taxid:85007) actinobacteria (taxid:201174), chlamydias (taxid:51291).

Various analyses place Alu repeats into 8 subfamilies having consensus sequences (GenBank; accession numbers U14567 - U14574). To correct for errors caused by Alu repeat artifacts in microbial sequences, microbial sequences were independently compared by the same methods to all 8 consensus Alu sequences and to 442 individual AluY sequences.

Often two comparisons were run simultaneously, one with humans excluded and one with humans included. Homologies identified were then sorted according to whether they involved teratogens, viruses or bacteria. Because homologies represent interspecies similarities, “Discontinuous Megablast” was used and the ten strongest homologies were measured in each run. Known teratogens included, HIV-1, N. gonorrhoeae, and N. meningitidis. Significant homology (indicated by the homology score) occurs when microbial and human DNA sequences have more similarity than would be expected by chance (E values). To minimize the chances for false positives, a cutoff for E values

When homologies were identified, the position in the target chromosomes were determined by entering the viral FASTA sequence into BLAT or into BLAST to determine its human genome location. Inserted sequences were also compared to Mus musculus GRCM38.p4 [GCF_000001635.24] chromosomes plus unplaced and unlocalized scaffolds (reference assembly in Annotation Release 106). The reference assembly set of RefSeq genomic top-level sequences (chromosomes, unplaced and unlocalized scaffolds) in a specific annotation run. Comparisons were also made to cDNAs based on 107,186 Reference Sequence (RefSeq) RNAs derived from the genome sequence with varying levels of transcript or protein homology support. Tests for contamination by vector sequences in these nontemplated sequences were also carried out with the BLAST program. Genes present in deleted segments were based on current genes identified in the UCSC Genome browser. Epigenomic modifications in associated histones in 9 model cell lines were also identified in the UCSC genome browser. In some cases, homology of inserted sequences to each other was tested using the Needleman and Wunsch algorithm.

Anatomical relationships in the nervous system show a close relationship and synchronization with structures essential for the development of immunity, circulation and protective enclosures. In chromosome segments deleted in neurologic disorders, genes essential for the nervous system, the immune system, circulation, brain-circulatory barriers and even bone structure are located close to each other on the same linear segment of a chromosome (Figure 2). Deletions at 4q34 in patient DGAP161 and at 19q12- 13.11 in patient DGAP125 are shown in Figure 2(a) and (b), respectively as examples that are representative of the other large chromosomal deletions. The genes within each deletion belong to each of the categories tested are color coded in the figure. These clustered arrangements are crucial because their deletion has been correlated with serious neurodevelopmental disorders [1]. In many cases, and alternatively spliced forms of the same gene may be used to encode for multiple functions that must be synchronized and coordinated among diverse cells. Multiple functions in different cell types are commonly found. Hormonal signaling represents a major control mechanism [11].

To investigate the chromosomal segment deletions that cause neurodevelopmental disorders, homologies to infection were tested in sequences within and flanking these deleted clusters. Strong homologies to infections were found interspersed throughout. To demonstrate the extent of these relationships, the deleted 4q34 chromosome segment (Patient DGAP161 [1]) was tested for homology to the top ten homologous microbes in 200 kb chunks. Figure 3 shows that stretches of homology to microorganisms are distributed throughout chromosome 4q34. Only 10 homologous microbes are shown for each 200 kb division, but there are up to hundreds, giving a total of many thousands of potentially homologous microorganisms throughout the 4q34 deletion.

A deeper analysis of the functions of genes within deleted chromosome segments predicts a predisposition to infection and correlates with symptoms of the developmental disorder. For example, deletion of chromosome 19q12-q13.11 occurs in patient DGAP125 (Figure 2). The deletion causes deficits in silencing provirus, repressing retrotransposons, responding to viruses, specifying immune cell lineages, and regulating apoptosis. The deleted 19q12-q13.11 band in DGAP125 had 76 matches to clostridium botulinum strain Mfbjuicb6. Many of these matches extended over 2800 bp at 72-73% homology with an E value of 0.0. The 5’ region included 43 of the 76 matches to clostridium covering about 2800 bp with 73% homology (E=0.0). The next million bp had another 33 matches to clostridium. There were also strong matches to Waddlia chondrophila and N. gonorrhoeae. Other shorter chromosomal anomalies are present in patient DGAP125. Stealth and HIV-1 teratogens and multiple other infections are homologous to these shorter anomalies (Figure 4).

The large deletion in 19q12-q13.11 makes patient DGAP125 especially susceptible to infections and the results are potentially devastating. The damage to defenses against infection associates with coordinated genes for control of breathing, synapse organization and plasticity, neural crest formation, and transmission of nerve impulses. There are simultaneous deficits in development of brain circulation and production of connective tissue and bone. Other chromosome deletions with clustered genes have deficits in neuronal accessory functions like those in DGAP125.

In some patients with neurodevelopmental disorders, robust evidence exists that the disease is caused by a truncated driver gene [1]. The gene changes identified as underlying phenotypic drivers of congenital neurologic diseases [1] were tested against existing evidence of their ability to act as epigenetic regulators and effectors of functions like those found in the chromosomal deletions. Table 1 shows that most of the pathogenic driver genes are epigenetic regulators. For example, gene products from FOXA1, PH21A, CHD7, and TCF4 mediate or regulate chromatin remodeling; ZBTB20 and GATA3 help regulate chromatin architecture; CDKL5 inactivates a histone deacetylase, WAC regulates histone ubiquitination; SOX5 encodes a histone demethylase; EHMT1 encodes a histone methyltransferase; MEF12C is associated with histone hypermethylation in schizophrenia; KDM6A encodes an epigenetic switch that specifies stem cell fate; EFTUD2 gene product associates with chromatin binding proteins as part of the spliceosome; SNRPN is required for epigenetic imprinting; TET1 is a methylation eraser. Short DNA sequences (less than 20,000 bp) deleted from families [3] are also likely to damage epigenetic regulators. Mutations also occurred in genes within topology associated chromatin regions and in insulators (e.g. CTCF, cohesin complexes).

Figure 4 graphically relates DNA sequences in all viruses and bacteria in the NCBI database to DNA sequences around breakage sites based on the study of Redin and colleagues [1]. In most cases, there is robust evidence that the chromosomal anomaly at breakage sites causes the neurodevelopmental disorder but there may also be cryptic rearrangements elsewhere [2,3,12]. The figure shows wide differences in the total homology scores of microbes vs humans in individual disorders. Patient DGAP154 has total homology scores of well over 50,000 while other patients (DGAP142, 172, and 173) are well below 1000.

Within a few hours of fertilization, global demethylation occurs and both maternal and paternal genomes reprogram to highly open chromatin [13]. Removal of these epigenetic marks allows a sudden increase in L1 retrotransposon expression which induces an innate immune response. Many of the chromosome anomalies studied contain L1- retrotransposon sequences. L1 transcriptional activity is controlled by DNA methylation and by piwi interacting RNA. Loss of epigenetic drivers can interfere with the function of these epigenetic control mechanisms (Table 1). Figure 5 summarizes this data and shows driver genes have epigenetic control functions and most of these functions impact chromatin 3D structure and multiple systems essential for neurodevelopment.

Because most identified driver genes of neurodevelopmental disorders are epigenetic regulators or effectors, the functions they control were compared to those found in chromosomal deletions that cause neurodevelopmental disorders. Virtually all pathogenic driver genes or chromosomal deletions in congenital neurologic disorders have strong relationships to effects on the immune system, angiogenesis, circulation and craniofacial development. Deletions in affected members of families with neurodevelopmental disorders are also consistent with this conclusion (Table 1).

In the 48 patients shown in Figure 4, multiple infections are candidates to cause the signs and symptoms in each individual (Table 2). 8 patients have growth retardation or “short stature” which can be caused by exposure to many infections in the second or third trimester. The absence, delay, or impairment of speech were found in 20 of 48 patients [1]. Multiple infections can cause these problems. For instance, HIV-1 causes white matter lesions associated with language impairments, and impaired fetal growth. There are nearly 50 matches to HIV-1 DNA in the chromosome anomalies of 35 patients.

Stealth viruses are frequent matches to chromosome anomalies with about 35 matching sequences. Stealth viruses are mostly derivatives of herpes viruses such as cytomegalovirus (CMV) that emerge in immunosuppressed patients or have other mechanisms to avoid recognition by the immune response. CMV is not a well-known teratogen, even though CMV is a major threat to infants. First trimester CMV infection can cause severe cerebral abnormalities followed by neurologic symptoms [14]. CMV is also a common cause of congenital deafness and can cause visual abnormalities. 27 of the 48 neurodevelopmental patients had hearing loss or deafness. Stealth virus 1 refers to Simian cytomegalovirus (strain Colburn) African green monkey cytomegalovirus, but closely related viruses with up to 95% sequence identity have been isolated from human patients. Herpes simplex virus is another stealth virus that directly infects the central nervous system. Herpes simplex can cause seizures (reported for 9 patients).

HTLV-1 is a retrovirus that causes an immune mediated disease of the nervous system affecting the spinal cord and peripheral nerves. Neural fetal cells are more susceptible to HTLV-1 infection than mature neurons.

Patient DGAP159 has the strongest homology to N. meningitidis (Figure 2). N. meningitidis causes bacterial meningitis and is a known cause of neurodevelopmental defects. Signs and symptoms in patient DGAP159 are consistent with residual effects of bacterial meningitis such as hearing loss, developmental delay, speech failure and visual problems.

Not only known teratogens, but almost all microbial DNA found in abnormal human chromosomes in congenital neurologic disorders can have profound effects on a fetus or neonate (Table 2). Figure 4 implies that any source of viral or microbial foreign DNA may contribute to developmental errors. Multiple matches may represent homologies to human DNA among stretches of viral and bacterial DNA. Homologies found appear independent of whether microbes can insert into human DNA. HIV, HPV16, HTLV-1, k. pneumoniae, ralstonia solanacearum, s. aureus, and stealth viruses are all found in at least 10 patients. 38 of 48 rearrangements include sequences that match one or more viruses, and 44 match bacterial sequences. In most chromosomal anomalies, multiple infections match a given anomalous DNA sequence. It is likely that most or all of these infections can interfere with neurodevelopment (Tables 1-2). Figure 4 includes endogenous retroviral sequences. Most of these endogenous sequences represent crippled infections that cannot travel between cells because they lack env genes needed to exit infected cells. However, HERV sequences are numerous and retrotransposons can still interfere with recombination and DNA repair within their home cell.

Effects of balanced translocations segregate only somewhat with the presence of neurodevelopmental disorders. These discrepancies emphasize the need to consider the genome in 3 dimensions [8]. Moreover, there may also be other more cryptic chromosome abnormalities and that whole genome sequencing of the family may be necessary [3]. Members of some families carry balanced chromosomal translocations but do not have signs and symptoms of neurodevelopmental disease. In three of four families with familial balanced chromosomal translocations, patient specific unbalanced deletions were also found [3] but the results did not overlap any database of human reference genomes. Unbalanced deletions were subjected to analysis for infection homology but inversions, inverted duplications, insertions and additional balanced translocations were also present. Unbalanced deletions in the three families were strongly homologous to infections or other foreign DNAs (Figure 6). In agreement with results in Table 1, critical genes linked to epigenetic modifications were among those interrupted by these short cryptic rearrangements.

De novo translocations t(11;22) have so far only been detected during spermatogenesis and not during other processes. These translocations have been attributed to palindromic structures that induce genomic instability [2]. To determine whether these structure might arise because microbial infection interferes with normal chromatin structures, homology testing of sequences in the recurrent breakpoint t(8;22)(q24.13;q11.21) [2] was conducted. Figure 7 shows strong homology to bacterial and viral sequences in the breakpoint region. Sequences from an unaffected mother carry a balanced translocation rearrangement [2] with homologies to more diverse microbes than are present in affected cases (Figure 7). This supports the idea that parts of a chromosome segment with homologies to infection have been deleted in affected family members.

Infection DNA occurs in neurodevelopmental disorders that undergo autosomal dominant inheritance so microbial DNA must be present in gametes from one parent. The mechanism proposed in Figure 8 is based on data showing significant homologies with microbial infections on multiple human chromosomes. Large amounts of foreign, infection DNA present during human meiosis with its many double strand breaks and the most active period of recombination produce defective gametes.

The resemblance of foreign microbial DNA to the host background DNA may be a major factor in selection of infection and in human ability to clear the infection. Only one rare defective gamete is shown in Figure 8 but the male generates four gametes during meiosis beginning at puberty. Only one gamete survives in the female because three polar bodies are generated. Both balanced and unbalanced translocations can give rise to chromosome anomalies such as deletions and insertions because infection DNA can insert itself (e.g. exogenous or endogenous retroviruses) or interfere with break repairs during meiosis. The presence of a preexisting balanced chromosomal translocation in the family disrupts normal chromatin structure, producing both affected and unaffected members [3]. The familial translocation increases the chances of generating a defective gamete during meiosis.

Genome rearrangements for patient data in Figure 4 [1] produced 1986 matches with E

The 1986 microbial homologues to human DNA near breakpoints involved in neurodevelopmental disorders were composed of 126 different viral or bacterial sequences. 24 different strains of Neisseria meningitidis in patient DGAP159 had 100% identity to human genomic rearrangements with total scores of 385 to 637 and expected values of 8e-13. The 25th N. meningitidis strain was 98% identical. Human matches included several 5’UTR like sequences, elongation factor alpha, isopentenyl pyrophosphate transferase-like protein, NADH dehydrogenases. Clostridium specifically matched sections of human ATPases, and ATP synthases, mitochondrial G elongation factor, and heat shock protein hsp70 (mRNA). A 1004 bp fragment of waddlia chondrophila occurred 32 times near neurodevelopmental breakpoints and had full-length 89% homology to human calcyclin binding protein (E=0.0). Waddlia chondrophila also had strong matches to human caspase recruitment domain and human cloned fragments. Another 55 of 126 viral / bacterial sequences were homologous to Alu sequences. Alu sequences are largely inactive retrotransposons, but it is possible that some of the homologies detected between humans and microbes are actually due to insertions from Alu or other repetitive sequences. Neuronal progenitors may support de novo retrotransposition in response to the environment or maternal factors [16]. Both non-Alu and Alu regions account for 90% (113 of 126) of the sequences homologous to microbes and present near breakpoints involved in human neurodevelopmental disorders.

To further test the possibility that some versions of these microbial sequences were sequencing artifacts or were contaminated by human genomes, microbial genomes represented in the Figures and other arbitrarily selected microbial genomes were tested for homology to human genomes. Results of these reversed-analyses make it unlikely that the human-microbial matches are errors or artifacts. Substantial similarities between partial and complete microbial DNAs and human DNAs were universal and helped develop the model in Figure 7.

Homologies between multiple strains of the same microbe and all human sequences were tested. Homologies to human sequences were found across multiple strains of the same microorganism (Table 3). In other cases, homology to some human gene product was found but there was not sufficient data available to compare multiple versions of the same microorganism. In these cases, the human gene was tested against all bacteria, and viruses. There were hundreds of significant matches. The complete clostridium botulinum genome had significant homology to human enzyme CDKL2 (86%) on chromosome 4. The region on chromosome 4: 579,587-75,632,298 spanned 52712 bp and was filled with over 50 Alu repeats, including multiple versions of AluS, AluY, and AluJ. Clostridium itself however did not have homology to Alu repeats. Clostridium also had significant homology with long stretches on other chromosomes including chromosomes 2, 3, 10, 15, 1, 5 with 74% homology over about 2853 bp E=0.0. Similarly, the full genome of waddlia chondrophila (2.1 million bp) was homologous to multiple human genes (Table 3).

Retroviral infections identified in Fig 1 are known to insert their sequences in human DNA or have other methods of infection and transmission, such as by inhibiting key protective mechanisms. For example, HIV-1 destroys helper T-cells after uptake by a specific receptor. Like other retroviruses, HIV-1 DNA becomes incorporated into host cells. HIV-1 and other retroviral insertions were found in the congenital abnormalities in neurodevelopmental disorders. HIV-1 isolate SC007 from India, partial genome (GenBank: KY713228.1) had 85% identity over 686 bases to an IgG2 lambda antibody and 82% homology to a 412 bp stretch of human DNA surrounding a rare NotI restriction site. HIV homologies were very commonly found among human sequences of neurodevelopmental disorders. To test for human sequence contaminants in these matching HIV sequences, an Alu homologous region of the HIV-1 genome (bp 7300 to 9000) in 28 different HIV-1 isolates in an HIV sequence compendium were compared. All 28 HIV sequences had matches to the same region of human DNA, with up to 98% identity to the human sequences. The best match was to a LINCRNA on chr11:74,929,289-74,929,412. In contrast, only one sequence of zika virus matched humans in a short segment of human-like DNA on the 3’ end. This homology was not found in 20 additional zika virus sequences so zika virus was not considered further.

It is almost impossible to completely exclude the possibility of sequencing artifacts or contamination of microbial sequences with human sequences. However rather than reflecting widespread, wholesale error due to human DNA contamination in many laboratories over many years, the vast majority of microbial homologies more likely suggest that infection sequences have been selected because they are homologous to regions of human DNA. This may be a driving force behind the much slower evolution of Alu DNA.

Long stretches of DNA in many infections match repetitive human DNA sequences which occur hundreds of thousands of times. Individual microorganisms also match nonrepetitive sequences. Human infections may be selected for and initially tolerated because of these matches. Infections can cause mutations by interfering with the most active period of recombination which occurs during the generation of gametes, an ongoing process in males. Infections such as exogenous or endogenous retroviruses are known to insert into DNA hotspots [8]. Human DNA sequences that closely match infection DNA generate chromosomal anomalies because similarities between microbial and human DNA cause interference with meiosis (Fig. 7). Thus, the genetic background of an individual may be a key factor in determining the susceptibility to infection and to the effects of infection. At the genetic level, this suggests selective pressures for infections to develop and use genes that are similar to human genes and to silence or mutate microbial genes that are immunogenic. Infection genomes evolve rapidly on transfer to a new host [17]. The presence of genes in infections that have long stretches of identity with human genes makes the infection more difficult to recognize as non-self. For example, there is no state of immunity to N. gonorrhoeae. One reason for this is that there are long stretches of N. gonorrhoeae DNA that are almost identical to human DNA. Alu sequences and other repetitive elements are thought to underlie some diseases by interfering with correct homologous recombination as in hereditary colon cancer [18] or abnormal splicing. Why this does not always occur is not well understood. The presence of infection DNA that is homologous to multiple, long stretches of human DNA may mask proper recombination sites and encourage this abnormal behavior.

Neurons and cells in the immune system are interacting systems, sensing and adapting to their common environment. These interactions prevent multiple pathological changes in many disorders [19]. Many genes that are implicated in neurodevelopmental diseases, reflect the strong relationship between the immune system and the nervous system. It was always possible to find functions within the immune system for genes involved in neurodevelopmental disorders (Figs 1 and 4). Damage to genes essential to prevent infection leads to more global developmental neurologic defects including intellectual disability. These microbial homologies include known teratogens. Analysis of mutations within clusters of genes deleted in neurodevelopmental disorders predict loss of brain-circulatory barriers, facilitating infections. Damage to cellular genes essential for the immune related function of autophagy may lead to abnormal pruning of neural connections during postnatal development.

Aggregated gene damage accounts for immune, circulatory, and structural deficits that accompany neurologic deficits. Other gene losses listed in Table 1 and in deleted chromosome segments (Figure 1) account for deficits in cardiac function, hearing, bone structure, pain perception, skull size, muscle tone and many other nonneurologic signs of neurodevelopmental disorders.

The arrangement of genes in clusters converging on the same biological process may simplify the regulation and coordination between neurons and other genes during neurodevelopment and neuroplasticity. Genes that are required for related functions, requiring coordinated regulation have been shown to be organized into individual topologically associated domains [20]. Neurons are intimately connected to chromatin architecture and the epigenetic landscape [21]. A disadvantage of the clustered arrangement of co-regulated accessory genes is that homology to microbial infections or other causes of chromosome anomalies anywhere in the cluster can then ruin complex critical neurological processes. Epigenetic regulators that affect multiple functions required by the same process make their mutation especially critical. Longer range developmental interactions in chromosome regions exacerbate the effects of infection. Mutations or deletions (Figure 1 and Table 1) show that this effect can occur in neurodevelopmental disorders. Functions that must be synchronized during neurodevelopment are grouped together on the same chromosome region and can be lost together. This is consistent with the idea that the organization of crossover chiasmata during meiosis allows the coordinated evolution and control of gene sets required by the same essential process.

Microbial DNA sequences are unlikely to be contaminants or sequencing artifacts. They are all found connected to human DNA in disease chromosomes; multiple sequences from different laboratories are all homologous to the same Alu sequence. Alu element-containing RNA polymerase II transcripts (AluRNAs) determine nucleolar structure and rRNA synthesis and may regulate nucleolar assembly as the cell cycle progresses and as the cell adapts to external signals [22]. HIV-1 integration occurs with some preference near or within Alu repeats, regardless of whether they occur in introns or exons [23].

The patients indicated in Fig. 3 have different disorders with different chromosome anomalies. This variability and the rarity of the anomalies makes these disorders difficult to study by conventional approaches. The techniques used here might eventually help predict the lifelong susceptibility to a given infection of an individual. However, the methods used suffer from the inability to absolutely identify one infection and to distinguish infection by one microorganism from multiple infections.

This research received no external funding but an Incentive award to BF was used for support.

This work is based on the outstanding DNA sequence information from investigators listed in the references and the author is grateful to them for making this information available. It is a pleasure to acknowledge the inspiration, challenge and stimulation provided by the Department of Biochemistry and Molecular Genetics and the College of Medicine at UIC.

The author declares no conflict of interest.