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Heterozygous FA2H mutations in autism spectrum disorders.

Scheid I, Maruani A, Huguet G, Leblond CS, Nygren G, Anckarsäter H, Beggiato A, Rastam M, Amsellem F, Gillberg IC, Elmaleh M, Leboyer M, Gillberg C, Betancur C, Coleman M, Hama H, Cook EH, Bourgeron T, Delorme R - BMC Med. Genet. (2013)

Bottom Line: Also, two rare non-synonymous mutations (R113W and R113Q) were reported.Although predictive models suggested that R113W should be a deleterious, we did not find that FA2H activity was affected by expression of the R113W mutation in cultured COS cells.While our results do not support a major role for FA2H coding variants in ASD, a screening of other genes related to myelin synthesis would allow us to better understand the role of non-neuronal elements in ASD susceptibility.

View Article: PubMed Central - HTML - PubMed

Affiliation: Human Genetics and Cognitive Functions, Institut Pasteur, Paris, France. richard.delorme@rdb.aphp.fr.

ABSTRACT

Background: Widespread abnormalities in white matter development are frequently reported in cases of autism spectrum disorders (ASD) and could be involved in the disconnectivity suggested in these disorders. Homozygous mutations in the gene coding for fatty-acid 2-hydroxylase (FA2H), an enzyme involved in myelin synthesis, are associated with complex leukodystrophies, but little is known about the functional impact of heterozygous FA2H mutations. We hypothesized that rare deleterious heterozygous mutations of FA2H might constitute risk factors for ASD.

Methods: We searched deleterious mutations affecting FA2H, by genotyping 1256 independent patients with ASD genotyped using Genome Wide SNP arrays, and also by sequencing in independent set of 186 subjects with ASD and 353 controls. We then explored the impact of the identified mutations by measuring FA2H enzymatic activity and expression, in transfected COS7 cells.

Results: One heterozygous deletion within 16q22.3-q23.1 including FA2H was observed in two siblings who share symptoms of autism and severe cognitive impairment, axial T2-FLAIR weighted MRI posterior periventricular white matter lesions. Also, two rare non-synonymous mutations (R113W and R113Q) were reported. Although predictive models suggested that R113W should be a deleterious, we did not find that FA2H activity was affected by expression of the R113W mutation in cultured COS cells.

Conclusions: While our results do not support a major role for FA2H coding variants in ASD, a screening of other genes related to myelin synthesis would allow us to better understand the role of non-neuronal elements in ASD susceptibility.

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Exploration of the somatic mosaicism in the Mother. (A) Results of NcoI digestion of FA2H PCR products from DNA extracted from lymphocytes and buccal cells. A NcoI site is created by the R113W mutation in exon 2. In both types of tissue tested, we found a lack of digestion in the father, partial digestion in both affected children and a low level of partial digestion in the mother. (B) Percentage of clones carrying the wild type (allele C, blue bar) and mutant alleles (allele T, red bar) as determined by cloning PCR products amplified from blood DNA of the clinically affected patient and his unaffected mother (Family 3).
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Figure 3: Exploration of the somatic mosaicism in the Mother. (A) Results of NcoI digestion of FA2H PCR products from DNA extracted from lymphocytes and buccal cells. A NcoI site is created by the R113W mutation in exon 2. In both types of tissue tested, we found a lack of digestion in the father, partial digestion in both affected children and a low level of partial digestion in the mother. (B) Percentage of clones carrying the wild type (allele C, blue bar) and mutant alleles (allele T, red bar) as determined by cloning PCR products amplified from blood DNA of the clinically affected patient and his unaffected mother (Family 3).

Mentions: All FA2H exons were directly sequenced in a subset of patients with ASD (n = 186) from the Paris study and in controls (n = 353). Two non-synonymous variations, R113Q and R113W, affecting the same amino-acid in exon 2, were identified in Families 2 and Family 3 respectively. No additional deleterious mutation was found in 353 controls and on the remaining allele of FA2H in either affected sib-pair from Family 1. In Family 2, R113Q was shared by both affected sibs. This variant was transmitted by the healthy mother, and detected in one of our controls (Figure 2). In silico, the R113Q variant was predicted to be benign whatever the model of prediction used (Polyphen-2/prediction: benign, PSIC score difference: 0.002; SNAP/prediction: neutral, reliability index: 3, expected accuracy 78%; SIFT/prediction: tolerated, SIFT score median: 0.67 to 2.09). In contrast, R113W was predicted to be damaging (Polyphen-2/prediction: damaging, PSIC score difference: 0.992; SNAP/prediction: non neutral, reliability index: 2, expected accuracy 70%; SIFT/prediction: damaging, SIFT score median: 0.02 to 2.09). This mutation was not observed in our control samples and in the 1000 genomes database (http://browser.1000genomes.org/) but was reported in 0.08%(11/12985) of the individuals referenced in the Exome Variant Server (http://evs.gs.washington.edu/EVS/). The analysis of the segregation reported that R113W was shared by both affected siblings and inherited from the mother in Family 3. The sequence chromatograph of the mother showed a reduced signal for the mutation (similar results were obtained when sequencing the forward and reverse strands). To determine if this reduced signal could be related to a somatic mosaicism, exon 2 containing a NcoI site created by the mutation was amplified from DNA extracted from lymphocytes and buccal swabs. In both tissues tested, digestion of PCR products amplified from family members showed a lack of digestion in the father, a partial digestion in both affected children and also a low level of partial digestion in the mother (Figure 3), suggesting partial expression. To determine the proportion of fragments in each PCR reactions harboring either the mutant (T) or the wild-type (C) allele, blood DNA was isolated from single plasmid clones, analyzed by restriction enzyme using NcoI and sequenced. Among the 165 clones sequenced in the mother, 113 (68.5%) contained the C allele vs. 52 (31.5%) contained the T allele (binomial test, z = 2.96, p = 0.002), suggesting a degree of somatic mosaicism. In her affected son, used as control, we were unable to detect any significant skewed distribution of the C allele (n = 91, 55.8%) vs. the T allele (n = 72, 44.2%) (binomial test, z = 0.91 , p = 0.18). The allelic distribution in the mother differed significantly from that observed in her son (X2 = 5.6, p = 0.02). These data suggest that the mother in Family 3 exhibits somatic mosaicism for the R113W mutation, which could explain the lack of cosegregation between the variant and autistic symptoms in the pedigree. However, since we were unable to obtain a second sample of blood genomic DNA of the mother, and we did not directly measure the allele bias by quantitative PCR, we could not confirm these results.


Heterozygous FA2H mutations in autism spectrum disorders.

Scheid I, Maruani A, Huguet G, Leblond CS, Nygren G, Anckarsäter H, Beggiato A, Rastam M, Amsellem F, Gillberg IC, Elmaleh M, Leboyer M, Gillberg C, Betancur C, Coleman M, Hama H, Cook EH, Bourgeron T, Delorme R - BMC Med. Genet. (2013)

Exploration of the somatic mosaicism in the Mother. (A) Results of NcoI digestion of FA2H PCR products from DNA extracted from lymphocytes and buccal cells. A NcoI site is created by the R113W mutation in exon 2. In both types of tissue tested, we found a lack of digestion in the father, partial digestion in both affected children and a low level of partial digestion in the mother. (B) Percentage of clones carrying the wild type (allele C, blue bar) and mutant alleles (allele T, red bar) as determined by cloning PCR products amplified from blood DNA of the clinically affected patient and his unaffected mother (Family 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Exploration of the somatic mosaicism in the Mother. (A) Results of NcoI digestion of FA2H PCR products from DNA extracted from lymphocytes and buccal cells. A NcoI site is created by the R113W mutation in exon 2. In both types of tissue tested, we found a lack of digestion in the father, partial digestion in both affected children and a low level of partial digestion in the mother. (B) Percentage of clones carrying the wild type (allele C, blue bar) and mutant alleles (allele T, red bar) as determined by cloning PCR products amplified from blood DNA of the clinically affected patient and his unaffected mother (Family 3).
Mentions: All FA2H exons were directly sequenced in a subset of patients with ASD (n = 186) from the Paris study and in controls (n = 353). Two non-synonymous variations, R113Q and R113W, affecting the same amino-acid in exon 2, were identified in Families 2 and Family 3 respectively. No additional deleterious mutation was found in 353 controls and on the remaining allele of FA2H in either affected sib-pair from Family 1. In Family 2, R113Q was shared by both affected sibs. This variant was transmitted by the healthy mother, and detected in one of our controls (Figure 2). In silico, the R113Q variant was predicted to be benign whatever the model of prediction used (Polyphen-2/prediction: benign, PSIC score difference: 0.002; SNAP/prediction: neutral, reliability index: 3, expected accuracy 78%; SIFT/prediction: tolerated, SIFT score median: 0.67 to 2.09). In contrast, R113W was predicted to be damaging (Polyphen-2/prediction: damaging, PSIC score difference: 0.992; SNAP/prediction: non neutral, reliability index: 2, expected accuracy 70%; SIFT/prediction: damaging, SIFT score median: 0.02 to 2.09). This mutation was not observed in our control samples and in the 1000 genomes database (http://browser.1000genomes.org/) but was reported in 0.08%(11/12985) of the individuals referenced in the Exome Variant Server (http://evs.gs.washington.edu/EVS/). The analysis of the segregation reported that R113W was shared by both affected siblings and inherited from the mother in Family 3. The sequence chromatograph of the mother showed a reduced signal for the mutation (similar results were obtained when sequencing the forward and reverse strands). To determine if this reduced signal could be related to a somatic mosaicism, exon 2 containing a NcoI site created by the mutation was amplified from DNA extracted from lymphocytes and buccal swabs. In both tissues tested, digestion of PCR products amplified from family members showed a lack of digestion in the father, a partial digestion in both affected children and also a low level of partial digestion in the mother (Figure 3), suggesting partial expression. To determine the proportion of fragments in each PCR reactions harboring either the mutant (T) or the wild-type (C) allele, blood DNA was isolated from single plasmid clones, analyzed by restriction enzyme using NcoI and sequenced. Among the 165 clones sequenced in the mother, 113 (68.5%) contained the C allele vs. 52 (31.5%) contained the T allele (binomial test, z = 2.96, p = 0.002), suggesting a degree of somatic mosaicism. In her affected son, used as control, we were unable to detect any significant skewed distribution of the C allele (n = 91, 55.8%) vs. the T allele (n = 72, 44.2%) (binomial test, z = 0.91 , p = 0.18). The allelic distribution in the mother differed significantly from that observed in her son (X2 = 5.6, p = 0.02). These data suggest that the mother in Family 3 exhibits somatic mosaicism for the R113W mutation, which could explain the lack of cosegregation between the variant and autistic symptoms in the pedigree. However, since we were unable to obtain a second sample of blood genomic DNA of the mother, and we did not directly measure the allele bias by quantitative PCR, we could not confirm these results.

Bottom Line: Also, two rare non-synonymous mutations (R113W and R113Q) were reported.Although predictive models suggested that R113W should be a deleterious, we did not find that FA2H activity was affected by expression of the R113W mutation in cultured COS cells.While our results do not support a major role for FA2H coding variants in ASD, a screening of other genes related to myelin synthesis would allow us to better understand the role of non-neuronal elements in ASD susceptibility.

View Article: PubMed Central - HTML - PubMed

Affiliation: Human Genetics and Cognitive Functions, Institut Pasteur, Paris, France. richard.delorme@rdb.aphp.fr.

ABSTRACT

Background: Widespread abnormalities in white matter development are frequently reported in cases of autism spectrum disorders (ASD) and could be involved in the disconnectivity suggested in these disorders. Homozygous mutations in the gene coding for fatty-acid 2-hydroxylase (FA2H), an enzyme involved in myelin synthesis, are associated with complex leukodystrophies, but little is known about the functional impact of heterozygous FA2H mutations. We hypothesized that rare deleterious heterozygous mutations of FA2H might constitute risk factors for ASD.

Methods: We searched deleterious mutations affecting FA2H, by genotyping 1256 independent patients with ASD genotyped using Genome Wide SNP arrays, and also by sequencing in independent set of 186 subjects with ASD and 353 controls. We then explored the impact of the identified mutations by measuring FA2H enzymatic activity and expression, in transfected COS7 cells.

Results: One heterozygous deletion within 16q22.3-q23.1 including FA2H was observed in two siblings who share symptoms of autism and severe cognitive impairment, axial T2-FLAIR weighted MRI posterior periventricular white matter lesions. Also, two rare non-synonymous mutations (R113W and R113Q) were reported. Although predictive models suggested that R113W should be a deleterious, we did not find that FA2H activity was affected by expression of the R113W mutation in cultured COS cells.

Conclusions: While our results do not support a major role for FA2H coding variants in ASD, a screening of other genes related to myelin synthesis would allow us to better understand the role of non-neuronal elements in ASD susceptibility.

Show MeSH
Related in: MedlinePlus