The authors selected families with one or more individuals affected by autism.

Among those families, about half have cousin marriages, which are referred to as consanguineous pedigrees.

A simplex family is one in which there is only one person with autism. A multiplex family has multiple people with autism.

To improve the quality of results, the authors chose consanguineous families (families that share a very recent ancestor), because of their high risk of having autistic children.

Researchers at Johns Hopkins University reasoned that families with multiple females with autism must have special genetic variants for autism that can be more easily identified than when comparing other groups.

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Refers to both the family tree and the ancestry of the subjects. In biology, pedigrees are represented with a standard set of symbols:

Phenomenon in which sections of the genome are repeated (2), deleted (1) or inverted (3). It has been shown that CNVs at specific locations increase the risk to develop autism.

Autism is associated with mental retardation about 70% of the time and that males are diagnosed more often.

Social class has no impact on the incidence of the disease, but there is not enough data to know if race or ethnicity influence the incidence of autism.

There is also no available data to support the idea that incidence of autism is changing over time.

Data supports the fact that autism is linked with several genomic regions and new regions are still being identified today.

A mutation is de novo when it appears for the first time in an individual, rather than being inherited. It is usually the result of a mutation in a germ cell of one of the parents, or a mutation that arises in the fertilized egg itself. It is also known as a new mutation.

Several research teams have worked separately to identify loci that could be responsible for autism. However, this has been difficult due to the high number of genes that could be involved and the high variability between affected individuals.

Autism is a "heterogeneous" disorder because not all autistic people have the same genetic anomalies. Autism is difficult to characterize not only because there are many different genes that can cause it, but also because autism sometimes results when several otherwise benign mutations come together in the genome of an individual.

See more in this Forbes article

Synapses-the electric signals sent by the nervous system-regulates the expression of certain genes.

Deletions in which the genetic information is missing on both chromosomes.

Locus (pl. Loci.) : The locus of a gene is its physical location on a specific chromosome.

Mapping consists of locating the genes on the chromosomes. Example for the gene BRCA:

The symptoms of autism vary in intensity along a spectrum. Unsurprisingly, the genetics of autistic patients also vary greatly, making it difficult to figure out what genes contribute to the disorder. Despite this, geneticists are still working toward decoding the genetics of autism. What kinds of mutations are present in autistic patients? How many are there? How can we improve methods to detect these errors? In this article, the authors present findings that move us closer to answering these questions.

This study was about mutations in the SLC9A6 gene, which are related with neurological diseases, such as X-linked mental retardation, microcephaly, epilepsy, and ataxia, a phenotype mimicking Angelman syndrome.

By using the experimental methods of linkage analysis and DNA sequencing they found out that a deletion in the SLC9A6 gene, encoding the Na(+)/H(+) exchanger NHE6, is basically responsible for these symptoms.

M. L. Jacquemont worked on the genetic heterogeneity of syndromic autism, more easily revealed through array-CGH. The author suggested that this method should be prioritized in order to diagnose autism. This study also revealed that patients with locus duplications are less affected that those presenting deletions. Finally, the importance of the chromosomal imbalance has no direct relation with the severity of the mental retardation.

Sebat and colleagues worked on the involvment of de novo Copy Number Variations in autism.

They showed that specific CNVs were mostly present in only one family among the tested group. They were also able to show the difference between simplex families, where autism is the result of spontaneous mutation and multiplex, where autism is most likely inherited.

mechanisms that involve the release of glutamate (a common neurotransmitter)

When families share a similar gene pool, it is likely that children will be homozygous for certain genes.

Objective: To analyse the mutations of NHE9 gene in patients with comorbid autism and epilepsy.

Methods: Sequencing of all exons and exons-intron boundaries in NHE9 gene

Results. It was found out that there is a heterozygous CGA to TGA transition in exon 1. This leads to transformation of arginine 423 to a stop codon and this change is quite similar to the one nonsense mutation in the Nhe1 gene.

Conclusion: both in NHE9 and Nhe1 genes nonsense mutations are observed in the last extracellular loop of NHE protein; NHE9 in the case of comorbid autism & epilepsy and Nhe1 in the case of slow-wave epilepsy.

In this research, the authors examined three affected males in the same family, all of whom exhibited characteristics as profound developmental delay, ataxia, hyperkinetic behaviour etc, which were suspects for Angelman syndrome, but relevant genetic examinations regarding this diagnosis had been negative.

They performed linkage analysis and DNA sequencing and they identified a deletion in the SLC9A6 gene encoding the Na+/H+ exchanger NHE6. Mutations in SLC9A6 cause X-linked mental retardation.

In this research, the authors worked on the description of the phenotype, genetic mapping, and identification of the defective gene in the swe mutant mouse.

This spontaneous mouse mutant, s low-w ave e pilepsy, (swe), is related to neurological syndrome, including ataxia, as well as a unique epilepsy phenotype consisting of 3/sec absence and tonic-clonic seizures.

It found to be located on chromosome 4 and identified as a null allele of NheI, which is ubiquitous, mediates the electroneutral 1:1 of Na+ and H+ and takes part in the regulation of the pH, cell volume and response to growth factors.

Swe mice were the first to model essential elements of human generalized absence epilepsy seizures and since it is characterised as null allele of Nhe1, it is the first disease-causing mutation identified in an Nhe gene.

Objective: To study the regulation of some autism-associated genes by neuronal activity.

Methods: They used genetic screens looking for genes regulated by neuronal activity or for targets of transcription factors induced by activity. Cultured rat hippocampal neutrons and complementary DNA (cDNAs) of tested genes were used in this method, as wells a parallel neuronal membrane depolarisation by elevated KCL. After gene expression was studied and compared during a period of 6 hours.

Results: c3orf58 gene expression was increased within 6 hours, as seen in Panel A, while by sequencing it found out to contain several evolutionarily conserved binding sites for MEF2, CREB and SRF (Panel B), things that implies that c3orf58 is a direct or indirect target of MEF2. PCDH10 gene showed more than doubled expression within 6 hours (Panel C). RNAi knock-down of NPAS4 was used; rat hippocampal cultures were transducer with either control virus (blue) or NPAS4 RNAi (red) and as seen in Panel D NHE9 expression was slightly altered.

Conclusion: Activity-regulated genes are related to homozygous deletions in autism patients, since either their coding sequence is mutated, as happens in case of c3orf58 gene or their conserved DNA sequences are affected, as happens in case of NHE9 and PCDH10.

In this article, the authors will focus on patients who inherited long deletions or additions from their parents. However, this is not the only approach to understanding the genetics of autism. Some look directly at the appearance of de novo SNP.

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Humans have chromosomes by pairs. An individual receives one chromosome from each parent. Therefore, he receives two copies of each gene. One says that an individual is homozygous for a certain gene when both of its chromosomes carry the same version of the gene.

Npas4 was found to cause activation of distinct programs of late-response genes in inhibitory and excitatory neurons. It's an activity-dependent transcription factor that regulates inhibitory synapse number and function in cell culture. It is also expressed in pyramidal neurons of the hippocampus where it promotes an increase in the number of inhibitory synapses on the cell soma and a decrease in the number of inhibitory synapses on the apical dendrites.

It is an exact test of the statistical significance of deviations from a theoretically expected distribution of observations from two categories.

It is used in many surveys, an example of how it works is given in the link below:

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Generally PCDH10 belongs to a proto cadherin protein family and it acts as a potential tumor suppressor protein, as the dysregulation of PCDH10 gene frequently existed in multiple human tumors.

PCDH10 is a transcriptional target of p53 and exhibits inhibitory roles in cancer cell motility and cell migration.

Autism: A per allele universe

The authors of this article compared their results with various studies of de novo CNVs and showed that the results obtained in this study are similar to the previous results obtained by Sebat and colleagues. Sebat and colleagues studied specifically proved that de novo CNVs detecting can be an interesting approach to detecting new genes linked with autism.

Figure 1 - Tab 1 : 'overview' The authors compared the genetic makeup of four members of a consanguineous simplex family. The analyses focuses here on a region on chromosome 3 presenting identity by descent (IBD), which means that two or more individuals inherited that region from a common ancestor without recombination. In this case, the IBD is only present in the affected individual. In this region, a 886kb deletion was identified and will be studied more closely.

Both the single nucleotide polymorphism array and BAC Comparative Genomic Hybridization arrays are used to detect the number of copies of a specific locus in a subject's DNA. This allows us to know whether the locus is present on one or both chromosomes of a subject.

To do this, control DNA and tested DNA are labeled with different fluorescent molecules of different colours (in the picture red for the control and green for the test). After denaturation, DNA will hybridise. If the sample DNA and the control DNA are identical, we will observe orange fluorescence. If the control DNA has deletions, the solution will appear red and if it gained regions, it will appear green.

The myocyte enhancer factor 2 (MEF2) transcription factor holds together the transcriptional circuits and controls cell differentiation and organogenesis. In adult tissues, Mef2 proteins are also reported to regulate the stress-response during cardiac hypertrophy and tissue remodeling in cardiac and skeletal muscle.

A strategy for down-regulation of expression of a gene by incorporating into the genome an antisense oligodeoxynucleotide or ribozyme sequence directed against the targeted gene.

A biological pathway, found in many eukaryotes, in which RNA molecules inhibit gene expression, usually by the destruction of certain mRNA molecules. This process is controlled by the RNA-induced silencing complex (RISC).

This procedure is also called co-suppression, post-transcriptional gene silencing (PTGS), or quelling.

https://www.youtube.com/watch?v=cK-OGB1_ELE&noredirect=1

This is an important and common experimental technique that is used to study the result of enhanced neuronal activity on alterations in gene expression.

The elevated KCL, which means the increase of extracellular potassium, has three mechanistic effects that result in a sustained depolarized state:

The normally present hyperpolarizing outflow of potassium (K+) is slowed down, while less hyperpolarizing outflow is equivalent to depolarization, since there is a depolarizing inflow of sodium (Na+) that was not also slowed down by an equivalent increase in intracellular sodium. The result is a change in the equilibrium potential of the cell.

The increase in the membrane potential is characterised as a slow depolarizasion and leads to the entrance of Na+ ions into the cell, via open sodium channels, resulting in further depolarization. The slow depolarization causes partial Na+ channel inactivation to occur as well. This prevents the neuron from triggering a full action potential.

As a result, the cell remains in a slightly depolarized state.

Neurons in the hippocampus, which is a major component of the human brain and those of other vertebrates. It is part of the limbic system and has a crucial role in the consolidation of information, from short-term memory to long-term memory and spatial navigation.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques.

In this article, a significant number of transcriptional regulators, which mediate activity-dependent gene transcription were identified.

This is interesting given the significant advances in understanding how the calcium-dependent activation of signalling cascades by extracellular stimuli takes part in the regulation of transcription-factor function.

A screen, also known as a genetic screen is a laboratory procedure used to create and detect a mutant organism and provide important information on gene function. In order to identify the function of an unknown gene, one strategy is to introduce general mutations into an organism and then study both the mutant and the control organisms in hopes of detecting a difference in their physical properties or phenotypes.It is used to identify and select for individuals who possess a phenotype of interest in a mutagenised population, so in this case autism-associated genes.

A new deletion was identified in patient AU-5101 removes the 5'region of the genes RNF8, which was present in patient AU-3101. This protein is a transcriptional activator. The deletion also encompasses the 3' region of another gene, and could have an impact on its expression.

Figure 2 Tab 1 : Overview The four figures show homozygous deletions identified using the SNP array. The upper part of each panel shows how many copies of each SNP the individual possesses, either none (deletion), one or two. The lower part of each panel shows the genes close to the deletions as well as the alignment between individuals.

Figure 1 - Tab 3 : 'Localisation of the deletion' Panel C zooms on the region of panel B that shows a copy number score of zero, which means that there is a homozygous deletion. The deletion is showed to be of 886kb in panel C. Both parents and the unaffected sibling have this deletion but on only one chromosome; this is shown by the copy number score of 1. On the contrary, the affected individual has a homologous deletion which is shown by the copy number score of 0.

The deletion affects the entire gene c3orf58, which codes for a protein present in the Golgi apparatus as well as the regulatory region of NHE9, which codes for an ion exchanger. This deletion can possibly affect the expression level of this protein.

The authors identified small deletions in the genome of the family in genes coding for various proteins that are highly expressed in the brain. However, it has not been proven that all these deletions play a role in the disease.

At the beginning of the transcription process, the genetic information of a strand of DNA is copied onto a new molecule, called messenger RNA (mRNA). The mRNA can then exit the nucleus of the cell to be translated into a protein.

This deletion was only present on one chromosome from each parent.

In this study, only two families presented overlapping loci. The region was identified in the study conducted by the International Molecular Genetic Study of Autism Consortium as a region where the highest LOD score was observed.

In consanguineous families, the authors observed that the proportion of affected women amongst the affected children was higher as well as the number of de novo CNVs was reduced.

This could mean that families with related parents present more recessive causes for autism. The authors will now try to determine what these causes are and where they are located on the genome.

After the experiments, the data was analyzed in order to determine the proportion of de novo copy number variants in each subject in comparison with a reference set of unaffected and affected subjects as well as the rates of de novo CNVs inherited from their parents.

The data showed that de novo CNVs were much less frequent in consanguineous families (both simplex and multiplex) and the amount of inherited CNVs is high.

The low rate of de novo copy number variant suggests that abnormalities in autistic patients' genomes comes from defective genes that were already present in the parents, rather than healthy genes that mutated before or shortly after fertilization.

The authors calculated the male to female ratio = (number of affected men)/(number of affected women) which is presented as ratio :1. This gives you how many men are affected for one affected women.

The logic was that there would be a difference in the ratio between consiguineous and non-consanguineous families if faulty genes were transmitted by autosomes.

In simplex families, only one person is affected by the disease. Multiplex families have more that one of their members affected by the disease.

Vorstman provides a review of much of the recent cytogenetic studies on autism. In these studies, researchers were able to identify abnormalities by looking at the shapes and sizes of chromosomes. From these observations, they were able to deduce if there were large-scale deletions, repetitions, or inversions in the chromosomes. A small percentage (about 3%) of autistic patients had mutations like this. Vorstman gathers these findings into one article and proposes regions scientists should focus on in future research.

Fisher's exact test is a statistical significance test, which is very useful in categorising data that result from classifying objects in two different ways; it is used for analysing contingency tables.

https://www.youtube.com/watch?v=jwkP_ERw9Ak

P. Szatmari and colleagues have shown that copy number variants (CNV) were risk factors and causal events for autism. However, they are not the only parameters to be taken into account : oligogenic inheritance - inheritance of genes which, in a small quantity, code major changes - would be a prevalent factor of autism.

An array is an orderly arrangement of samples where known and unknown DNA samples are matched according to base pairing rules.

In this experimental setup, the cDNA derived from the mRNA of known genes is immobilised. The expression pattern is then compared to the expression pattern of a gene responsible for a disease.

https://www.youtube.com/watch?v=pWk_zBpKt_w

The research team led by De Silva studied the genetic factors associated with ADHD. One of the identified genes was SLC9A9 and they discovered that this gene codes for a sodium/hydrogen ion exchanger that is expressed in the brain, but also in the heart and the skeletal muscles.

Hoodfar and Teebi studied the link between inbreeding and the prevalence of different genomic abnormalities. In this study, the amount of autosomal recessive disorders was more than doubled in consanguineous families.

Figure 1 Tab 2 : 'Identifying homozygous regions' In panel A, the authors present the genotyping data of each individual to study wether each SNP is homozygous or heterozygous. You can see that the affected individual presents a large region of homozygous SNPs which is also the 74cM IBD.

A variation of a single nucleotide in a DNA sequence. When less than 1% of a population carries the nucleotide at this position, this variation is classified as a SNP. Some SNPs are associated with certain diseases.

Reminder : Homozygosity Mapping Collaborative for Autism

DNA is a macromolecule forming a long double helical shape and containing the “blueprint” of an organism. It gives the instructions for the production of all the proteins of the organism. Each long portion of DNA is called a chromosome.

Figure 2 Tab 2 : 'panel A' the Authors found a large deletion of 321kb in a cluster of genes coding for protocadherin proteins. These proteins are involved in cell adhesion. It is also worth noting that this deletion is close to PCDH10, where a deletion was identified in AU-3101.

In this article, R. Canitano emphasizes the link between autism and epilepsy, showing that seizures are more frequent when mental retardation is associated with autism. The rate of comorbidity - cohabitation between two diseases, autism and epilepsy here - is estimated at 20-25%, meaning that this amount of autistic people is victim of seizures. This link has to be taken into account when considering therapy.

Figure 2 Tab 3 : 'Panel B and C' In two different individuals, deletions in the 5' noncoding region of genes were identified.A small deletion of 150kb was found in patient AU-5801 which deletes the upstream noncoding region of a gene coding for Contactin-3. This protein mediates cell surface interactions during the development of the nervous system.

A 47kb deletion was identified in patient AU-8101 in the 5' noncoding regions of SCN7A, which codes for a sodium channel protein.

Deletions in the 5' noncoding regions could impact the expression levels of these proteins.

E. Fombonne has provided a review of epidemiological surveys on autism, in order to draw general conclusions. He concluded that autism is associated with mental retardation in about 70% of the cases and is overrepresented amongst males. He also showed that social class has no impact on the incidence of the disease. He also considered that available surveys did not allow to conclude that race or ethnicity influence the incidence of autism. Finally, no data could support the idea of secular evolution in the incidence of the disease.

Other genes the authors identified are involved in how cells move proteins around. Cells usually do this by packing proteins into endosomes (small spheres made out of membrane) to send proteins to specific places.

A second big deletion was identified on the chromosome. This codes for a protein that allows for neuron, and more specifically axon, growth.

This deletion completely removes a protein-coding gene called c3orf58, and a large section of possible regulatory regions of another gene called NHE9. SK

The authors focused on one pedigree in this paragraph: the family of patient AU-3101. A first deletion removes an entire gene, coding for a protein present in the Golgi Apparatus, as well an upstream region of the gene coding for NHE9, an ion exchanger.

The authors noticed that in some cases, there were deletions of certain long sequences present on both chromosomes of the affected individuals. These huge deletions could cause symptoms of autism in certain cases.

The abnormalities in autistic patient's chromosomes were not shared between families. This suggests that autism can have many different genetic origins.

The authors observed that the proportion of women affected doubles in consanguineous multiplex compared to the whole HMCA and is almost tripled compared to simplex families.

Risch and colleagues studied the genetic makeup of subjects affected by autism.

They showed that autism is most likely caused by multiple genes and identified several genes that could play a part in this disease in different individuals.

This study was about the regulation of specific transcription factors by neuronal activity. They found out, by using techniques as mass spectrometry and chromatin immunopreuritation, that synaptic activity determines the expression of a set of neural gene products.

In this study also, a large nuclear protein, Cabin 1 (calcineurin-binding protein), was found out to be q cqlcium-regulated depressor of MEF2 activity.

This study was about a spontaneous mouse mutant, called slow-wave epilepsy (swe), which is related to several neurological syndromes, like ataxia, as well as about a unique epilepsy phenotype, that is characterized by 3/sec absence and tonic-clonic seizures.

swe was fine-mapped on Chromosome 4 and identified as a null allele of Nhe1, which is the Na+/H+ exchanger, a ubiquitous, and acts as an integral membrane protein involved in pH regulation. It removes intracellular acid, exchanging a proton for an extracellular sodium ion.

A stop codon (or termination codon) is a nucleotide triplet, which stops the synthesis of a protein molecule. It's placed within messenger RNA and signals a termination of translation into proteins.

This termination is taking place due to binding release factors, which cause the ribosomal subunits to disassociate, releasing the amino acid chain.

The set of all messenger RNA transcripts in the considered cell.

The authors found that large deletions of genes that are regulated by neuronal activity were associated with autism symptoms. This is an interesting finding, because autism symptoms often develop later in development.

Some mutations can affect a neuron's ability to adhere to other neurons. This difficulty can result in autism symptoms.