Carla A. Brandon

Digital three-dimensional photogrammetry: evaluation of anthropometric precision and accuracy using a Genex 3D camera system.

Objective To determine the precision and accuracy of facial anthropometric measurements obtained through digital three-dimensional (3D) photogrammetry. Design Nineteen standard craniofacial measurements were repeatedly obtained on 20 subjects by two independent observers, using calipers and 3D photos (obtained with a Genex 3D camera system), both with and without facial landmarks labeled. Four different precision estimates were then calculated and compared statistically across techniques. In addition, mean measurements from 3D photos were compared statistically with those from direct anthropometry. Results In terms of measurement precision, the 3D photos were clearly better than direct anthropometry. In almost all cases, the 3D photo with landmarks labeled had the highest overall precision. In addition, labeling landmarks prior to taking measurements improved precision, regardless of method. Good congruence was observed between means derived from the 3D photos and direct anthropometry. Statistically significant differences were noted for seven measurements; however, the magnitude of these differences was often clinically insignificant (< 2 mm). Conclusions Digital 3D photogrammetry with the Genex camera system is sufficiently precise and accurate for the anthropometric needs of most medical and craniofacial research designs.

Mutations in BMP4 Are Associated with Subepithelial, Microform, and Overt Cleft Lip

Cleft lip with or without cleft palate (CL/P) is a complex trait with evidence that the clinical spectrum includes both microform and subepithelial lip defects. We identified missense and nonsense mutations in the BMP4 gene in 1 of 30 cases of microform clefts, 2 of 87 cases with subepithelial defects in the orbicularis oris muscle (OOM), 5 of 968 cases of overt CL/P, and 0 of 529 controls. These results provide confirmation that microforms and subepithelial OOM defects are part of the spectrum of CL/P and should be considered during clinical evaluation of families with clefts. Furthermore, we suggest a role for BMP4 in wound healing.

Possible Association of Amelogenin to High Caries Experience in a Guatemalan-Mayan Population

There is evidence for a genetic component in caries susceptibility, but the disease is greatly influenced by environmental factors, which are extremely difficult to control in humans. For the present study, we used DNA samples collected from 110 unrelated, non-cleft individuals older than 12 years of age from Tiquisate, Guatemala: a population with similar cultural, dietary and hygiene habits, similar access to the dentist and fluoride exposure. Forty-four individuals were designated ‘very low caries experience’ (DMFT ≤2), and 66 were designated ‘higher caries experience’ (DMFT ≧3). Single-nucleotide polymorphism markers were genotyped in selected candidate genes (ameloblastin, amelogenin, enamelin, tuftelin-1, and tuftelin interacting protein 11) that influence enamel formation. Having at least one copy of the rare amelogenin marker allele was associated with increased age-adjusted caries experience. This association was stronger in individuals with higher DMFT (DMFT ≧20; p = 0.0000001). Our results suggest that variation in amelogenin may contribute to caries susceptibility in the population studied. The approach of comparing individuals with extremely distinct caries experiences could be valuable for decreasing the potential influence of environmental factors on genetic studies of caries.

Abundant expression of myosin heavy-chain IIB RNA in a subset of human masseter muscle fibres

Type IIB fast fibres are typically demonstrated in human skeletal muscle by histochemical staining for the ATPase activity of myosin heavy-chain (MyHC) isoforms. However, the monoclonal antibody specific for the mammalian IIB isoform does not detect MyHC IIB protein in man and MyHC IIX RNA is found in histochemically identified IIB fibres, suggesting that the IIB protein isoform may not be present in man; if this is not so, jaw-closing muscles, which express a diversity of isoforms, are likely candidates for their presence. ATPase histochemistry, immunohistochemistry polyacrylamide gel electrophoresis and in situ hybridization, which included a MyHC IIB-specific mRNA riboprobe, were used to compare the composition and RNA expression of MyHC isoforms in a human jaw-closing muscle, the masseter, an upper limb muscle, the triceps, an abdominal muscle, the external oblique, and a lower limb muscle, the gastrocnemius. The external oblique contained a mixture of histochemically defined type I, IIA and IIB fibres distributed in a mosaic pattern, while the triceps and gastrocnemius contained only type I and IIA fibres. Typical of limb muscle fibres, the MyHC I-specific mRNA probes hybridized with histochemically defined type I fibres, the IIA-specific probes with type IIA fibres and the IIX-specific probes with type IIB fibres. The MyHC IIB mRNA probe hybridized only with a few histochemically defined type I fibres in the sample from the external oblique; in addition to this IIB message, these fibres also expressed RNAs for MyHC I, IIA and IIX. MyHC IIB RNA was abundantly expressed in histochemical and immunohistochemical type IIA fibres of the masseter, together with transcripts for IIA and in some cases IIX. No MyHC IIB protein was detected in fibres and extracts of either the external oblique or masseter by immunohistochemistry, immunoblotting and electrophoresis. Thus, IIB RNA, but not protein, was found in the fibres of two different human skeletal muscles. It is believed this is the first report of the substantial expression of IIB mRNA in man as demonstrated in a subset of masseter fibres, but rarely in limb muscle, and in only a few fibres of the external oblique. These findings provide further evidence for the complexity of myosin gene expression, especially in jaw-closing muscles.

Orbicularis oris muscle defects as an expanded phenotypic feature in nonsyndromic cleft lip with or without cleft palate

Nonsyndromic cleft lip ± cleft palate is a complex disease with a wide phenotypic spectrum; occult defects of the superior orbicularis oris muscle may represent the mildest subclinical form of the lip portion of the phenotype. This study used high‐resolution ultrasonography to compare the frequency of discontinuities in the OO muscle in 525 unaffected relatives of individuals with nonsyndromic cleft lip  ±  cleft palate versus 257 unaffected controls. OO muscle discontinuities were observed in 54 (10.3%) of the non‐cleft relatives, compared to 15 (5.8%) of the controls—a statistically significant increase (P = 0.04). Male relatives had a significantly higher rate of discontinuities than male controls (12.0% vs. 3.2%; P = 0.01); female relatives also had a higher rate of discontinuities than female controls, but the increase was not statistically significant (8.9% vs. 7.4%; P = 0.56). These data confirm the hypothesis that subepithelial OO muscle defects are a mild manifestation of the cleft lip phenotype. Identification of subepithelial OO muscle defects may be important in a clinical setting, as a means of providing more accurate recurrence risk estimates to relatives in cleft families. Furthermore, the expansion of the cleft lip  ±  cleft palate phenotypic spectrum should improve the power of genetic studies.

Enamel Formation Genes Influence Enamel Microhardness Before and After Cariogenic Challenge

There is evidence for a genetic component in caries susceptibility, and studies in humans have suggested that variation in enamel formation genes may contribute to caries. For the present study, we used DNA samples collected from 1,831 individuals from various population data sets. Single nucleotide polymorphism markers were genotyped in selected genes (ameloblastin, amelogenin, enamelin, tuftelin, and tuftelin interacting protein 11) that influence enamel formation. Allele and genotype frequencies were compared between groups with distinct caries experience. Associations with caries experience can be detected but they are not necessarily replicated in all population groups and the most expressive results was for a marker in AMELX (p = 0.0007). To help interpret these results, we evaluated if enamel microhardness changes under simulated cariogenic challenges are associated with genetic variations in these same genes. After creating an artificial caries lesion, associations could be seen between genetic variation in TUFT1 (p = 0.006) and TUIP11 (p = 0.0006) with enamel microhardness. Our results suggest that the influence of genetic variation of enamel formation genes may influence the dynamic interactions between the enamel surface and the oral cavity.

Rethinking isolated cleft palate: Evidence of occult lip defects in a subset of cases†

Emerging research suggests that subepithelial defects of the upper lip musculature are part of the phenotypic spectrum of cleft lip and/or palate (CL/P) and may represent an occult, subclinical manifestation of the anomaly. The present study investigates whether similar occult lip defects are present in individuals affected with isolated cleft palate (CP). To this end, upper lip ultrasounds of 33 CP cases (12 males, 21 females) were evaluated retrospectively for the presence of discontinuities (i.e., breaks) within the orbicularis oris muscle (OOM). In four CP cases (2 males, 2 females), distinct discontinuities of the OOM were identified. Of the remaining CP individuals, 23 demonstrated normal lip morphology on ultrasound (7 males, 16 females), while, in 6 cases (3 males, 3 females), a definitive evaluation was not possible. As CP and CL/P are traditionally thought to be etiologically distinct, these findings raise the possibility that some CP cases may be misclassified. Such diagnostic errors could have important implications for recurrence risk estimation and studies aimed at discovering etiology.

UNLOADED SHORTENING VELOCITY AND MYOSIN HEAVY CHAIN VARIATIONS IN HUMAN LARYNGEAL MUSCLE FIBERS

Myosin description in human laryngeal muscles is incomplete, but evidence suggests the presence of type I, IIA, IIX, and tonic myosin heavy chain (MHC) fibers. This study describes the unloaded shortening velocity (V0) of chemically skinned laryngeal muscle fibers measured by the slack test method in relation to MHC content. Skeletal fibers from human laryngeal and limb muscle biopsy specimens were obtained for determination of V0, and subsequently, glycerol–sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used to determine the MHC isoform content. The fibers from human limb muscle had shortening speeds similar to those in previous reports on human skeletal fibers. Type I, IIA, and IIX fibers of laryngeal muscle had shortening speeds similar to those of fibers from limb muscle, but laryngeal fibers with heterogeneous MHC expression had a wide range of shortening speeds, some being nearly twice as fast as limb fibers. In addition, MHC isoform bands from human extraocular muscle comigrated with some bands from laryngeal muscle — a finding suggesting that extraocular myosin may also be expressed.

Staining of Human Thyroarytenoid Muscle with Myosin Antibodies Reveals Some Unique Extrafusal Fibers, but no Muscle Spindles

This study describes the myosin composition of extrafusal and intrafusal muscle fibers found in the human thyroarytenoid (TA) and sternohyoid (control) muscles. We sought to determine the presence of muscle spindles in the TA muscle, and to identify unusual extrafusal fiber types, using the commonly accepted approach of tissue staining with myosin isoform specific antibodies. Extrafusal fibers are organized into motor units, which subsequently produce muscle movement, whereas intrafusal fibers compose muscle spindles, the primary stretch receptor that provides afferent (feed back) information to the nervous system for regulation of motor unit length and tonicity. Immunohistochemical identification of muscle spindles was confirmed in sternohyoid, but not in TA samples; however, some extrafusal fibers contained tonic myosin. These results indicate that human TA muscle functions similar to some mammalian extraocular muscle, performing unloaded (non-weight bearing) contractions without afferent information from native muscle spindles.

Whorl patterns on the lower lip are associated with nonsyndromic cleft lip with or without cleft palate.

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a common birth defect due to both genetic and environmental factors. Whorl lip print patterns are circular grooves on the central upper lip and/or the left and right lower lip. To determine if whorls are more common in families with CL/P than in controls, the Pittsburgh Orofacial Cleft Study collected lip prints from over 450 subjects, that is, individuals with CL/P, their relatives, and unrelated controls—from the U.S., Argentina, and Hungary. Using a narrow definition of lower‐lip whorl, the frequency of whorls in the U.S. sample was significantly elevated in cleft individuals and their family members, compared to unrelated controls (14.8% and 13.2% vs. 2.3%; P = 0.003 and 0.001, respectively). Whorls were more frequent in CL/P families from Argentina than in CL/P families from the U.S. or Hungary. If these results are confirmed, whorl lip print patterns could be part of an expanded phenotypic spectrum of nonsyndromic CL/P. As such, they may eventually be useful in a clinical setting, allowing recurrence risk calculations to incorporate individual phenotypic information in addition to family history data.