We proposed that ASD might be associated with a distinctive facial phenotype and hypothesized that we might be able to identify subgroups within ASD based on facial phenotypes. Using sophisticated facial phenotyping based on three-dimensional stereophotogrammetric imaging and advanced statistical analyses, we developed a research methodology that allowed us to test and garner support for both hypotheses. In addition, we maximized our chances of success by selecting a relatively homogeneous ASD population that included only boys of self-reported Caucasian ethnicity, with essential autism, defined as having no discernible dysmorphology or microcephaly and within a limited age range.
We found that essential autism in boys is associated with a distinctive facial phenotype characterized by an increased breadth of the mouth, orbits and upper face, combined with a flattened nasal bridge and reduced height of the philtrum and maxillary region. This facial phenotype is similar to the one we recognized clinically (JHM) and may be the "beautiful face" mentioned by Kanner [58
]. Embryologically, this facial phenotype is indicative of a perturbation of the FNP. It can be explained by reduction in the superoinferior dimension of the midline structures derived from the FNP, an increase in the subnasal portions of the FNP and a concomitant increase in the breadth of the upper face.
The common facial phenotype described by Rodier and colleagues [25
] includes a reduced interpupillary distance with no difference in intercanthic distance. In contrast, in our present study, we found a narrowing of the intercanthic distance, or mild hypotelorism. These findings are complementary in that we found an overall decrease in intercanthic distance, which potentially translates to decreased interpupillary distance, although we did not directly measure that distance. Our findings are also in line with those of Hammond et al
]. However, the sample included in the Hammond et al
] study consisted of a group of boys, ages 2 to 18 years from families with at least two affected family members. Our study extends the findings of both Rodier and colleagues [25
] and Hammond et al
] by quantifying precisely localized differences and variations in facial phenotypes in a homogeneous group of boys with essential autism.
Our findings further extend previous work in that we have discovered two subgroups of boys with ASD who displayed unique facial phenotypes, which correspond to distinct clinical phenotypes, compared to both the majority of boys with ASD and TD boys. Subgroup 1 displays decreased height of the facial midline and increased breadth of the mouth as well as the length and height of the chin. These regions of the face develop primarily from the FNP and midline portions of the MAND prominences of the embryonic face. Subgroup 2 displays increased breadth of the upper face in combination with decreased height of the philtrum. Both of these regions develop from the embryonic FNP.
The results of the tests of our hypotheses indicate that boys with ASD have an altered developmental pattern of the structures derived from the embryonic FNP and the MAND. It is well-documented that the developing FNP is derived from localized, specific cell populations under patterned genetic control. A number of developmental genes have been implicated in patterning the outgrowth of FNP, including FGF8, SHH and BMP2.
Neural crest cells that ultimately make up the FNP migrate over the forebrain to become encased within the neural ectoderm of the forebrain and the facial ectoderm [19
plays a chemoattractive role in neural crest cell migration [20
]. Signals from the forebrain neuroectoderm are essential for the survival of the neural crest cells of the FNP [30
], including SHH-dependent signaling from forebrain [21
]. Other studies have shown that SHH provides a key signal in regulating facial neural crest cell survival and patterning [22
] and regulates BMP2
expression in the middle and upper face [18
]. Furthermore, it has been shown in an animal model that decreased SHH signaling leads to narrowing of the FNP and hypotelorism [59
]. The MAXs and LNPs are responsive to WNT signaling [32
]. WNT signaling leads to elevated cell proliferation and migration of neural crest cells [32
]. No midline structures are responsive to WNT.
Correct patterning and development of the forebrain also requires a balance of these genetic signaling factors [18
], including SHH and the FGF, BMP and WNT families [20
is expressed in the facial ectoderm and neuroectoderm at various developmental stages and acts synergistically with FGF8
in both face and brain development [31
expression in facial ectoderm affects the expression of FGF8
in the brain [30
]. Likewise, expression of FGF8
by the forebrain is stimulated by the presence of neural crest cells [61
It is clear that development of the face and brain is an interactive process, both anatomically and genetically. Gene expression studies have shown that facial and neural tube development are intimately interrelated. Altered gene expression patterns are associated with alterations in face and brain development. The reverse is also true: Altered face and brain development is associated with alterations in gene expression. We know that the brain is altered in people with ASD (reviewed in [62
]), and the results of our present study show that the face is also affected. However, the sequence of events leading to these differences is unclear and may differ among the various subgroups described herein.
There is evidence derived from genetic studies implicating the developmental genes that control the patterning of the FNP and forebrain which causes autism. The SHH
gene, though not identified by autism linkage or association studies, is functionally related to the Patched
), which is a strong autism candidate gene. Investigators who conducted CNV studies [63
] first identified PTCHD1
gene microdeletions and missense mutations in males with ASD [64
signaling repression is relieved when SHH binds to PTCHD1. BMP
is part of the functional face and brain patterning network interacting with SHH and FGF8
to maintain brain and face patterning. Bakrania et al
] evaluated gene expression in embryos and demonstrated cotemporal and cospatial expression of BMP4
signaling genes. It is expected that sophisticated brain and/or face functional transcriptome studies may be useful in further linking genes involved in simultaneous face and brain development.
The clinical and behavioral differences that we identified between subgroup 1 and subgroup 2 boys support our hypothesis that the subgroups are biologically and etiologically distinctive. Subgroup 1 appears to be more severely autistic, with only 8% diagnosed with Asperger syndrome compared to 60% in subgroup 2, and has higher SCQ lifetime scores than subgroup 2 (25.3 vs 17.7). The percentage of IQ scores less than 70 were higher in subgroup 1 than in the other groups: 50% on the FSIQ and 45% on the NVIQ, compared to only 20% on both the FSIQ and NVIQ (one of five) in subgroup 2. and 15% FSIQ and 12% NVIQ in the ASD remainder group. Verbal IQ scores were also lower in subgroup 1 but did not reach statistical significance. Subgroup 1 also displayed several features predictive of poor outcome, including a higher risk for seizures and increased incidence of language regression at ASD onset. The observation of significantly higher SRS scores in mothers of boys in subgroup 1 is interesting but unexplained at this time.
By contrast, subgroup 2 appears to be aligned more with an Asperger syndrome diagnosis, which was made in 60% of subgroup 2 participants compared to only 8% in subgroup 1 and 32% in the remainder. Lifetime SCQ scores in subgroup 2 were also lower (17.7) than those in subgroup 1 (25.9) and the remainder (20.6). Consistent with the Asperger syndrome diagnosis, boys in subgroup 2 spoke their first words significantly earlier than boys in subgroup 1 and the remainder, and they were significantly more likely to be macrocephalic (80% vs 17%) compared to subgroup 1. Though IQ score differences did not reach significance within this small subgroup, boys in subgroup 2 had consistently higher FSIQ, NVIQ and VIQ scores than boys in subgroup 1.
Though we maximized our study outcomes by using a relatively homogeneous group of boys of Caucasian ancestry with essential autism within a narrow age range, the subject group was imperfect in a number of ways. Though 56% of the boys had participated in the SSC, not every participant completed the entire test battery, which slightly decreased the number of subjects who could be analyzed statistically for some comparisons. However, all of the four boys without VIQ scores and the one boy without a FSIQ score clustered morphologically within the main group and not within either subgroup. Thus our findings are highly unlikely to be affected by the small number of missing data points. In addition, the SSC population is biased toward a higher-functioning group of boys, which tended to shift the cognitive and outcome curves. The higher-functioning population recruited from the SSC may, however, have helped delineate subgroup 2 by increasing the proportion of subjects with Asperger syndrome. In addition, the number of boys in each subgroup was small. One additional observation is that in subgroup 2, which contained only five boys, one of the five was an outlier with significantly lower scores on IQ, language and outcome measures. This suggests that in future studies of larger numbers of subjects, we may find that subgroup 2 will be dissected into subgroups 2A and 2B. Finally, although one of the strengths of our study is that it comprised a homogeneous group of boys, that is, narrow age range, single sex, limited ethnic diversity and diagnosis made by a single clinician, it remains to be seen whether our findings will be consistent in a more heterogeneous population.