While it is unlikely that every alteration identified in this study is causative of autism, several variants appear especially interesting in their relationship to this disease. Specifically, there are two alterations which may alter the amino acid sequence sufficiently to interfere with protein function. The first is the R23M alteration in MBD3. This alteration falls within the methyl-CpG-binding domain of the protein and replaces the basic arginine with a nonpolar methionine. While this amino acid is not completely conserved in the other members of the MBD family, another basic amino acid, lysine, is found in the same position for the MeCP2, MBD1 and MBD2 proteins. An alteration of the charge at this position could interfere with the function of the MBD domain. It is also interesting that the variation is inherited from the maternal grandmother through a mother presenting with an anxiety disorder to two affected half brothers. This leaves open the possibility that there is a sex-specific effect for this variation.
The second variation of interest is E314fsX316 within MBD4. This maternally inherited allele is predicted to cause a frameshift at amino acid 314, encode two aberrant amino acids, and then a stop codon, resulting in a prematurely truncated protein. The mutant protein would contain the MBD domain, but the DNA glycosylase domain would be absent. While it is likely that much of the abnormal RNA would be removed by nonsense-mediated decay [56
], it is conceivable that any truncated protein generated with an intact MBD domain may be able to interfere in the normal function of all of the MBD proteins. Indeed, Bader and colleagues recently demonstrated in vitro that a MBD4 protein truncated at amino acid 313 could inhibit the glycosylase activity of the wild-type MBD4 protein as well as uracil DNA glycosylase [57
]. An alternative scenario is that an irregular phenotype could simply result from haploinsufficiency of the wild-type protein. This change warrants further investigation.
There are additional examples of amino acid changes detected in the MBD proteins that could result in functional changes. There are three nonsynonymous changes, MBD1 R147K, MBD1 K558N, and MBD4 E346K, which either remove or create lysine residues. Lysine is an amino acid subject to dramatic posttranslational modifications including methylation, acetylation, sumoylation, ubiquitination, and neddylation [58
]. The MBD proteins have demonstrated that they are susceptible to such modifications [59
]. Altering this key amino acid could dramatically influence the function of a protein, affect protein regulation, or interfere with protein–protein interactions. Another example of a variation that may affect posttranslational modification is S342P in MBD4. The disruption of a serine could remove a potential site of phosphorylation or glycosylation. Likewise for the creation of a serine site, as occurs in the MBD4 N467S variation within the glycosylase domain. Other variants which alter the polarity and charge of the residues, as seen in missense mutations in MBD1 and MBD4, could interfere with a binding site on the protein or cause misfolding.
It is also possible that some of the detected MBD variations could result in RNA processing errors by destroying splice sites, creating cryptic splice sites, or even changing the ratios of alternative isoforms by eliminating or generating binding sites for splicing factors (Supplementary Table 3
). One example of this is the MBD1
intronic variant c.1585-12T>C, just 12 bp upstream of exon 14. The alteration causes a change in a highly conserved nucleotide, potentially breaking a consensus site for the SRp55 splicing factor and creating new binding sites.
Surprisingly, there appears to be a higher occurrence of variations with potential effects on amino acid function within the African-American autistic patients as compared to Caucasian patients. African-Americans comprise only 31 of the 226 (13.7%) families participating in this study, yet these families represent a disproportionate number of those with nonsynonymous alterations (24%). This could be due, in part, to the higher genetic diversity found in African populations [63
]. Furthermore, missense changes that were most abundant varied between the ethnicities, with the MBD4 A273T (rs10342) change being the most common for Caucasian families and the MBD4 S342P (rs2307289) alteration being predominantly found in African-American families. These results are consistent with the HapMap data for these known SNPs in the AA and CA populations. In addition, four of the five deletions identified in this study occurred only in African-American families, three of which were not identified in any control individuals. The remaining MBD3
GCG deletion was identified in both AA and CA affected and controls while the MBD3
GAG deletion was found exclusively in AA affected and controls, suggesting that both deletions are most likely not associated with autism. Nonetheless, these racial differences in variant frequencies add to previous findings in which African-Americans with autism showed subtle differences in phenotype and association to SNPs in GABAergic genes [65
In the families with nonsynonymous mutations, psychiatric disorders were reported (on family history interview) in first degree relatives who carry a variation. This is consistent with previous reports of an increased prevalence of neuropsychiatric disorders including schizophrenia, depression, and anxiety in the parents of autistic children [67
]. Inheritance of MBD variants from parents diagnosed with psychiatric disorders was found in families numbers 7978, 7974, 7941, and 7785 (). Moreover, two families have additional offspring who carry the same variant found in their affected sibling and has psychiatric problems without a diagnosis of autism. The sister in family no. 7978 was diagnosed with anxiety and speech delay, and a brother in family no. 7710 also presented with delayed speech (). This lends support to the idea that the variants identified here could contribute to autism and other psychological defects. Indeed, some variants were identified in both affected and unaffected individuals within a family and multiplex families were not always concordant for the variant being identified. In some instances, as outlined above, the variants appear in people negative for autism, but demonstrating clinical characteristic found in autism. Such is the case for family no. 7974 where a mother with anxiety/panic disorder passes along the MBD3 R23M variant to two autistic half brothers. The variation in phenotypic presentation may also be related to incomplete penetrance of genetic factors. This can be demonstrated by the fact that there is an increase in the concordance of autism in monozygotic twins as compared to dizygotic twins, but there is not 100% concordance in MZ twins [4
]. Therefore, although genetics plays a strong role in autism etiology, it is not the sole component influencing presentation of disease. Lack of perfect concordance should not be used as a measure to rule out causal mutations within complex diseases. Other factors such as environmental or epigenetic events may also contribute to the intricacy of autism.
It is intriguing that we observed clinical similarities in select patients to the Rett syndrome phenotype. While, by definition, Rett syndrome and autism overlap clinically, there are features common to Rett syndrome which are less frequent in the general autistic population. For example, regression is found in most classical manifestations of Rett, but only in a minority of autistic individuals [1
]. Yet, we identified eight cases with a regression in language, in some cases accompanied by losses in social responsiveness or play skills. Furthermore, select individuals were reported to have irregular breathing, unusual gait, and seizures, all traits are commonly found in Rett patients. Also of note were two individuals identified with unusual midline movements, reminiscent of the classic Rett feature where girls clasp their hands together at the center of their body [15
]. Perhaps, these patients are presenting clinically in a manner more similar to Rett individuals due to the alterations identified in this study within the MBD genes. It is also interesting that the MBD1 R147K alteration identified in family no. 7801 was found in the two more severely affected of three autistic brothers as well as an unaffected sister. This could suggest that this alteration results in a sex-specific effect that may contribute to the autism phenotype without being the sole causative factor.
While there is mounting evidence from human and mouse studies implicating MBD genes in autism, few patients have been identified with alterations in MBD1
, MBD2, MBD3
, and MBD4
]. This seeming inconsistency may be clarified by evaluating the murine phenotypes. While the Mbd1−/−
mouse exhibits deficiencies in a wide range of social behaviors, it is able to live an almost normal life span [48
]. In contrast, Mecp2−/−
mice have a severe phenotype that is usually lethal by 10 weeks of age [39
]. Observing the relatively mild phenotype of the Mbd1
null mice in relation to the Mecp2
null mice suggests that there may be an ascertainment bias in identifying patients with recognized MECP2
mutations may lead to such a mild phenotype that they rarely present as clinical abnormalities [48
In summary, we identified genetic alterations at 46 unique loci in the MBD genes. Twenty-one were previously recognized SNPs, while 25 represent novel variations (five insertion/deletions and 20 SNPs). Although the majority of alterations were synonymous or noncoding variants, we identified ten nonsynonymous changes in MBD1, MBD3, and MBD4, the deletion of a single amino acid in MBD3, and a frameshift mutation in MBD4 that is predicted to truncate almost half of the protein. These results suggest that rare variants in these genes may contribute to autism susceptibility. Further studies utilizing larger cohorts will be required to determine the potential contribution of these genes to the autism clinical spectrum, but evidence to date indicates that the entire MBD family may play a role in autism etiology.