Candidate gene study of HOXB1 in autism spectrum disorder

Genetic contributions to autism have received strong support from family and twin studies [1, 2]. A relatively small number of major loci was initially predicted to explain the disease in the majority of affected individuals [2]. However, the number of common genetic variants conferring vulnerability to autism has grown well beyond the initial expectations, in addition to several rare variants identified to this date, each explaining the disease in a very small number of patients [[3‐5]. The genetic underpinnings of autism spectrum disorders have thus proven to be far more complex than expected, likely due to genetic heterogeneity, epistasis and gene-environment interactions [3‐5].

Several lines of evidence have demonstrated altered prenatal neurodevelopment as central to autism pathogenesis. Abnormal neurodevelopment possibly occurring in the first trimester of pregnancy best accounts for the microscopic alterations shown by post-mortem neuroanatomical studies of the brains of autistic patients [6, 7]. Phenotypic evidence also supports a prenatal aetiology, as fine motor abnormalities are detectable at 4-6 months of age or even at birth in children later developing autism [8]. Finally, a prenatal time window as early as days 20-25 post-fertilization appears crucial to autism's aetiology, since exposure to thalidomide during pregnancy leads to autism only if occurring within this limited developmental interval [9]. Genes encoding proteins involved in early neural development could thus encompass polymorphisms or mutations contributing to the disease process.

The HOXB1 gene, located on human chr 17q21.32, is a member of the HOX gene family of homeobox transcription factors and critically involved in the development of the brain stem. HOXB1 gene expression is limited to rhombomere 4 and to neural crest cells migrating out of this region, resulting in a gross reduction or complete loss of the facial motor nucleus in HOXB1 mutant mice [10, 11]. A similar loss of facial motor neurons has been described in one autistic brain [12]. HOXB1 gene expression occurs very early in mouse development (E8.5-E9.5) [10, 11], at a time, interestingly, overlapping with the window of maximal prenatal sensitivity in rodent models of autism [13]. HOXB1 gene expression is also strongly up-regulated by HOXA1[14] and the two genes indeed synergize in patterning hindbrain structures, cranial nerves and pharyngeal arches, so that double-mutant mice display prominent malformations while single mutants suffer much milder abnormalities [14‐16]. A gene × gene interaction between HOXB1 and HOXA1 gene variants was initially proposed to contribute to autism [17]. In our sample, the HOXA1 c.218A>G [His73Arg] polymorphism was significantly associated with autism, although we found an association with the A218 and not the G218 allele [18], in contrast to the original study [17]. Moreover, HOXA1 c.218A>G alleles were found to significantly influence head growth rates, and not final head size, both in autistic patients and in typically developing children [18, 19]. The latter result is especially interesting, considering that approximately 20% of autistic patients consistently show macrocephaly and may represent an endophenotypic subgroup possibly sharing common pathophysiological underpinnings [20, 21]. Following the initial positive study [17], five studies subsequently reported negative association findings using HOXB1 gene markers [22‐26]. However, the sample sizes assessed in all of these studies were too small to detect moderate-size effects and rare variants of potential clinical relevance (see Discussion). Furthermore, only two of these studies apparently performed mutational searches by DNA sequencing [22, 23]. The present study was thus undertaken to screen a larger sample of autistic patients, unaffected controls and nuclear families for HOXB1 gene mutations, rare variants and common polymorphisms, either causing the disorder, conferring autism vulnerability, influencing the clinical expression of the disease, or modulating cranial growth rates by themselves or in interaction with HOXA1 gene variants.

The present study describes three rare (IVS1+63G>A, V234V and S291N) and three common (c.82ins9, Q103H and A150A) variants in the HOXB1 locus of a total sample of 269 ASD patients. No pathogenetic de novo mutation was found as all these gene variants were inherited from one of the parents and at least two are present in other population samples available in public databases. Also, HOXB1 common variants do not seem to play major roles in autism pathogenesis, although minor contributions cannot be excluded due to sample size limitations (see below). At the phenotypic level, HOXB1 alleles appear to modulate head growth rates to a much lesser extent compared to the HOXA1 c.218A>G SNP (Figure 4, compare panels E versus A and B) [18, 19]. In reference to cranial circumference, we also find no evidence of gene-gene interaction, since both HOXA1 and HOXB1 influence head growth independently of each other (Figure 4F). Finally, preliminary analyses involving the subgroup of patients characterized also with the ADI-R suggest that HOXB1 alleles may influence stereotypic behaviours. Given the relatively large number of clinical variables assessed for association with HOXB1 alleles, this finding survives correction for repeated measures only applying the 'relaxed' criteria, namely those accounting for the non-independence of several variables which are significantly cross-correlated in our sample [38].

In interpreting these results, three limitations of our study design should be considered: (a) we have not specifically assessed for the presence of CNVs encompassing the HOXB1 locus in our sample - from a methodological standpoint, DHPLC is not able to distinguish true homozygosity from deletion of one allele; (b) despite employing a significantly larger sample size, compared to previously published reports [17, 22‐26], our study is still underpowered for common variants in the allelic frequency range of the INS and T309 alleles (see Table 5 at allelic frequencies of 0.2-0.3) and for highly penetrant de novo mutations with dominant effects and allelic frequency below 0.02 (Table 6 at an allelic frequency of 0.01); and (c) we cannot evaluate the possible differences in mutation burden at this locus between patients and controls because mutational analysis was performed only among our patient sample.

A novel and interesting finding is represented by the location and pattern of inheritance of rare variants. In particular, rs35115415 [c.702G>A (V234V)] and c.872_873delinsAA (S291N) are located in genomic regions which have been evolutionarily conserved from Tetraodon nigroviridis and Caenorhabditis elegans all the way up to Homo sapiens (Figures 2 and 3). This homeobox domain is probably under high selective pressure and disruptive mutations are probably incompatible with life. Mutations or chromosomal rearrangements involving HOX genes and compatible with life are in most cases highly disruptive in animals and in humans, resulting in abnormal limb formation, severe neurological syndromes, leukaemias, or solid tumours [40, 41]. In reference to unaffected individuals, publically available databases report one large chorodial neovascularization (CNV; duplication/deletion) involving HOXB1, among many other genes, found in two of the 270 individuals of the HapMap collection (see http://​projects.​tcag.​ca/​variation/​, variation 4040, landmarks chr17:43,957,672... 44,191,836) [42], indicating that the human genome may tolerate some deletions and duplications involving the entire HOXB1 locus. However, the HOXB1 gene segments hosting rs35115415 [c.702G>A (V234V)] and c.872_873delinsAA (S291N) may possibly accommodate only the less disruptive and non-pathogenic gene variants. Another interesting issue is the parental transmission to the same autistic proband of two distinct rare variants, one transmitted from each parent (Figure 1). Instances where rare variants (single nucleotides and/or CNVs) have no causal effect in heterozygous carriers but are pathogenic in a state of compound heterozygosity are increasingly recognized as a possible cause of autism [43]. However, the rare HOXB1 variants described in the present study do not appear pathogenic, as it is biologically implausible that in family T24_IT, for example, the convergence of the intronic variant IVS1+63G>A and the conserved variant 702G>A (V234V) in the same individual may bear dramatic functional consequences. Within the framework of the 'compound heterozygosity' hypothesis, a similar phenomenon should also be occurring in at least one other autism-causing locus. The a priori probability of the compound heterozygosity occurs by chance at the HoxB1 locus can be estimated at 1.8 × 10^-5 and 9.0 × 10^-6 for families T24_IT and USA85, respectively. The probability of this same phenomenon occurring by chance at two separate loci in the same family is extraordinarily low, unless boosted by a deficit in genome maintenance mechanisms. Some [44, 45], though not all studies [46], support the hypothesis that a small, yet a sizable minority of autism families may display an increased degree of genomic instability. This hypothesis, which is clearly not addressed in the present report, will be the object of further investigation

In summary, our data indicate that: (a) HOXB1 gene variants, either rare or common, do not exert large or moderate effects on affection status in autism spectrum disorders, while minor contributions cannot be excluded due to sample size limitations; (b) modulatory effects on head growth rates are compatible with the developmental roles of this homeobox gene: and (c) HOXB1 could influence the clinical autistic phenotype in the area of stereotypic behaviours.

The peculiar coincidence of two distinct rare variants inherited by the autistic probands from both parents in two families raises interest in dysfunctional genome maintenance mechanisms in a subgroup of ASD patients.