Analysis of candidate genes on chromosome 2 in oral cleft case-parent trios from three populations.

Isolated oral clefts, including cleft lip with/without cleft palate (CL/P) and cleft palate (CP), have a complex and heterogeneous etiology. Case-parent trios from three populations were used to study genes spanning chromosome 2, where single nucleotide polymorphic (SNP) markers were analyzed individually and as haplotypes. Case-parent trios from three populations (74 from Maryland, 64 from Singapore and 95 from Taiwan) were genotyped for 962 SNPs in 104 genes on chromosome 2, including two well-recognized candidate genes: TGFA and SATB2. Individual SNPs and haplotypes (in sliding windows of 2-5 SNPs) were used to test for linkage and disequilibrium separately in CL/P and CP trios. A novel candidate gene (ZNF533) showed consistent evidence of linkage and disequilibrium in all three populations for both CL/P and CP. SNPs in key regions of ZNF533 showed considerable variability in estimated genotypic odds ratios and their significance, suggesting allelic heterogeneity. Haplotype frequencies for regions of ZNF533 were estimated and used to partition genetic variance into among-and within-population components. Wright's fixation index, a measure of genetic diversity, showed little difference between Singapore and Taiwan compared with Maryland. The tensin-1 gene (TNS1) also showed evidence of linkage and disequilibrium among both CL/P and CP trios in all three populations, albeit at a lower level of significance. Additional genes (VAX2, GLI2, ZHFX1B on 2p; WNT6-WNT10A and COL4A3-COL4A4 on 2q) showed consistent evidence of linkage and disequilibrium only among CL/P trios in all three populations, and TGFA showed significant evidence in two of three populations.

Testing candidate genes for non-syndromic oral clefts using a case-parent trio design.

Markers in five candidate genes were examined on 269 case-parent trios ascertained through a child with an isolated, non-syndromic oral cleft (cleft lip, CL; cleft palate, CP; or cleft lip and palate, CLP). Cases and their parents were ascertained through treatment centers in Maryland. Markers at two of the five candidate genes, transforming growth factor beta3 (TGFbeta3) and MSX1, showed consistent evidence of linkage and disequilibrium due to linkage using several statistical tests (e.g., the global chi-square for TGFbeta3 was 21.1 with 12 df, P = 0.03; that for MSX1 was 8.7 with 3 df, P = 0.03). There was little evidence of heterogeneity in the role of TGFbeta3 between different types of oral clefts, but MSX1 did yield marginal evidence for such heterogeneity. MSX1 also showed evidence for interaction between infant's genotype and maternal smoking, giving a likelihood ratio test for heterogeneity between smoker and non-smoker mothers of 7.16 (2 df, P = 0.03). Using a conditional logistic model to test for gene-gene interaction showed no evidence of interaction between TGFbeta3 and MSX1, with both seeming to contribute independently to risk of isolated, non-syndromic oral clefts.

A case-control study of nonsyndromic oral clefts in Maryland.

PURPOSE: Isolated, nonsyndromic oral clefts cases (n = 171) and unaffected controls (n = 182) were used to identify both genetic and environmental risk factors. METHODS: Infants born in Maryland between 1992 to 1998 with an isolated, nonsyndromic oral cleft [cleft lip (CL), cleft lip and palate (CLP), or cleft palate (CP)] were recruited and exposure plus family history data were collected. Controls were unaffected infants. DNA was collected from all cases and their parents, plus controls. RESULTS: No statistically significant association was found between any of the following: maternal smoking, vitamin use, urinary tract infection, or recreational drug use in either univariate analysis or after adjusting for maternal age and education. More control mothers reported alcohol use during the critical time period of pregnancy (one month before conception through the first trimester) as compared to case mothers. There was a 10-fold increase in risk to siblings of cases as compared to siblings of controls. Markers at four candidate genes were examined: transforming growth factor alpha (TGF alpha), transforming growth factor beta 3 (TGF beta 3), MSX1, and BCL3. Only MSX1 showed significant differences in allele frequencies between CP cases and controls. MSX1 also showed significant evidence of linkage disequilibrium with a susceptibility gene controlling risk for CP. CONCLUSION: Most environmental risk factors examined here gave little evidence of association with risk to isolated, nonsyndromic oral clefts, although any alcohol consumption seemed protective. MSX1 showed evidence of linkage disequilibrium in both case-control and case-parent trio analysis.

An assistive device for the treatment of positional plagiocephaly.

An assistive device (AD) was used to treat 122 infants with moderate-to-severe positional plagiocephaly. Clinical evaluations indicate that the AD provided the most benefit when applied to 2- to 8-month-old children. Our results suggest that the AD may join molding helmets and physical therapy as a treatment for moderate-to-severe positional plagiocephaly in infants under the age of 1 year.

Three-dimensional morphological analysis of isolated metopic synostosis.

Morphological differences were quantified in three-dimensions among individuals with untreated isolated metopic synostosis and between those individuals and similar aged-matched normal dry skulls to test two hypotheses: first, that the dysmorphology is a self-correcting condition; and second, that a lack of vertical growth of the skull produces this dysmorphology. Three-dimensional (3D) coordinates were recorded for 22 craniofacial landmarks from CT scans of 15 metopic patients, ranging from 5- to 32-months-old, and of four normal dry skulls, ranging in age from 6- to 36-months-old. The patient population was diagnosed with isolated metopic synostosis at The Johns Hopkins Medical Institutions in Baltimore, Maryland or Children's Hospital in St. Louis, Missouri. Comparisons between the metopic age groups indicate that the trigonocephalic phenotype worsens through time. Between 5 and 14 months, the neurocranium displays an increase in vertical growth. This was followed by a lack of vertical growth between 14 and 32 months. The face displays a lack of vertical growth from 5 to 14 months and an increase in vertical growth after 14 months. Comparisons between the metopic age groups and the normal skulls indicate that the trigonocephalic head is taller superoinferiorly and longer anteroposteriorly. Relative to the normal phenotype, the inferior temporal region in the metopic phenotype is narrow. These findings enabled the rejection of both hypotheses and localized form differences between normal and metopic phenotypes. Based on these results, we suggest that the trigonocephalic phenotype worsens with age and the amount of vertical growth that produces the trigonocephalic phenotype varies throughout growth with respect to location within the skull and age.

Improved free musculocutaneous flap survival with induction of heat shock protein.

The cellular response to a wide variety of stresses results in the synthesis of a family of stress response proteins termed heat shock proteins. Recent studies have demonstrated that heat shock proteins produced in response to an initial stress seem to protect against subsequent unrelated stresses. Importantly, hyperthermia-induced heat shock proteins provided protection from ischemia/reperfusion injury in several organ transplantation models. We hypothesized that free musculocutaneous flap survival could be improved by enhancing the flap's tolerance to relative ischemia by the prior induction of heat shock proteins. Accordingly, we determined the heat shock protein response in skin and muscle after systemic or local heating and examined the effect on free musculocutaneous flap survival in a rat model. Free musculocutaneous flaps incorporating thigh adductor muscles and a 2 x 6-cm2 skin paddle were transplanted to the ipsilateral groin in three groups of male Wistar rats. Systemically heated rats (n = 6) were anesthetized and incubated for 30 minutes at 42 degrees C 6 hours before free musculocutaneous tissue transfer. Locally heated rats (n = 6) were anesthetized, and their donor site anterior thigh was placed for 30 minutes on a heating block set at 44 degrees C 6 hours before free tissue transfer. Control rats (n = 5) did not have heating pretreatment but underwent identical anesthesia. Animals were sacrificed on postoperative day 3, at which time skin loss (cm2) and muscle viability, quantified by nitroblue tetrazolium staining time, were assessed in a blinded fashion. The skin and muscle from the free flap were analyzed for HSP72 mRNA and protein using quantitative Northern and Western blot techniques. All free musculocutaneous flaps were viable. However, the locally and systemically heated rats demonstrated a marked improvement of skin survival, which correlated with increased skin levels of HSP72. There were no differences in nitroblue tetrazolium muscle staining times or muscle levels of HSP72 among the three groups. These findings suggest that prior heat-induced heat shock proteins result in improvement in musculocutaneous flap survival, which may have direct clinical applications, especially in high-risk patients.

Testing for interaction between maternal smoking and TGFA genotype among oral cleft cases born in Maryland 1992-1996.

OBJECTIVE: Infants born in Maryland between June 1992 and June 1996 were used in a case-control study of nonsyndromic oral clefts to test for effects of maternal smoking and a polymorphic genetic marker at the transforming growth factor alpha (TGFA) locus, both of which have been reported to be risk factors for these common birth defects. DESIGN AND SETTING: Cases were infants with an oral cleft ascertained through three comprehensive treatment centers, with additional ascertainment through a registry of birth defects maintained by the Maryland Health Department. Controls were healthy infants. Medical history information on infants and mothers were collected, along with DNA samples. PATIENTS, PARTICIPANTS: Among 286 cases contacted (72% ascertainment), there were 192 nonsyndromic isolated oral clefts (106 M; 86 F) available for this case-control study. MAIN OUTCOME MEASURES: The largest group of 149 Caucasian nonsyndromic cases and 86 controls was used to test for association with maternal smoking and genotype at the Taq1 polymorphism in TGFA. RESULTS: While this modest sample had limited statistical power to detect gene-environment interaction, there was a significant marginal increase in risk of having an oral cleft if the mother smoked (odds ratio = 1.75, 95% CI = 1.01 to 3.02). We could not demonstrate statistical interaction between maternal smoking and TGFA genotype in this study, however, and the observed increase in the C2 allele among cases was not statistically significant. CONCLUSIONS: We could not confirm either the reported association between oral clefts and TGFA genotype or its interaction with maternal smoking. However, these data do show an increased risk if the mother smoked during pregnancy, and this effect was greatest among infants with a bilateral cleft and no close family history of clefts.

Lambdoid synostosis and occipital plagiocephaly: clinical decision rules for surgical intervention.

Lambdoid craniosynostosis has been regarded as one of the least common categories of premature fusion of the cranial sutures, yet reports have suggested the incidence may be increasing. To guide treatment decisions, the authors describe a set of rules based on radiographic indicators and clinical assessment in the child. Experience suggests that children can have abnormal-appearing cranial sutures with normal neurological status and normal-appearing sutures with neurological deficits or marked cerebral compression. Early evaluation and follow-up treatment is essential for children with suspected craniosynostosis.

Application of transmission disequilibrium tests to nonsyndromic oral clefts: including candidate genes and environmental exposures in the models.

Extensive epidemiological and genetic studies of the cause of oral clefts have demonstrated strong familial aggregation but have failed to yield definitive evidence of any single genetic mechanism. We used the transmission/disequilibrium test (TDT) to investigate the relationship between oral clefts and markers associated with five candidate genes by utilizing 160 parent-offspring trios. Conditional logistic regression models extended the TDT to include covariates as effect modifiers, thus permitting tests for gene-environment interactions. For four of these candidates [transforming growth factor alpha (TGFA), transforming growth factor beta 3 (TGFB3), retinoic acid receptor (RARA), and the proto-oncogene BCL3], we detected modestly elevated odds ratios for the transmission of one marker allele to cleft probands when all the trios were analyzed together. These odds ratios increased when information on type of cleft, race, family history, or maternal smoking were incorporated as effect modifiers. We detected significant interaction between maternal smoking and the transmission of alleles for markers near TGFA and TGFB3; excess transmission of allele 3 at BCL3 was most significant among cleft lip probands; and the odds ratios for transmission of alleles at D19S178 and THRA1 were significant when ethnic group was included in the model. We suggest that utilizing an analytical strategy that allows for stratification of data and incorporating environmental effects into a single analysis may be more effective for detecting genes of small effect.

Rigid fixation of internal orbital fractures.

When large portions of the internal orbit are destroyed (two to four walls), standard bone-grafting techniques for immediate and late orbital reconstruction may not yield predictable eye position. Critical bone support is most often deficient inferomedially. CT analysis of orbital volume in cases where eye position was unsatisfactory reveals that displacement of bone grafts is one mechanism of the unsatisfactory result. Other mechanisms include undercorrection and bone-graft resorption. In order to minimize postoperative bone-graft displacement, titanium implants were used to span large defects in the internal orbit to provide a platform for bone-graft support. Twenty-six implants were placed in immediate and 12 were placed in late orbital reconstructions. More reliable bone-graft position resulted. Two late infections have occurred resulting in implant removal in a 3-year period.

The redistribution of collagen in expanded pig skin.

Silicone tissue expanders were inserted subcutaneously in the buttocks of nine young pigs and gradually inflated to maximum capacity over 5 weeks. On the control side the expanders were left uninflated. Island buttock flaps were then raised, the expanders removed and the flaps spread into the same sites for 10 days. The tissue was harvested. Area measurements and full thickness skin biopsies were taken 10 days after flap inset in order to study the changes in collagen composition and isotypes in the skin layers. Ten days after inset of the flap the expanded skin had a mean 47% increase in surface area, was 9% thinner (from surface to implant), mostly due to thinning of the subcutaneous zone, but was not significantly different in water content, relative to the control skin. The expanded skin had a significant 9.3% increase (p less than 0.01, t test) in collagen content of the dermis. The relative proportions of Types I and III were not significantly changed by skin expansion in either the dermal/epidermal or subcutaneous/capsular zones. It is speculated that tensile factors during expansion stimulate the biosynthetic activity and/or mitotic activity of fibroblasts in the dermis to produce this gain in collagen in the expanded compared with unexpanded tissue.

Rigid stabilization of sagittal fractures of the maxilla and palate.

Rigid stabilization of sagittal fractures of the palate is described that utilizes plate and screw fixation in the palatal vault. Accurate reduction of facial width is obtained, and stability is significantly enhanced. An existing laceration or a longitudinal incision in the palatal mucoperiosteum provides exposure for maxillary adaption plate application. The transpalatal reduction should be supplemented by fixation at the piriform aperture, the zygomaticomaxillary and nasomaxillary buttresses, and by the use of an arch bar. Since slower bone healing may be observed following palatoalveolar fractures, the occlusion must be observed for deviation throughout a full 16-week period even though early motion and soft diet are permitted. Removal of the plate and screws in the roof of the mouth is sometimes required and utilizes local anesthesia.

Techniques for improved visibility and lid protection in orbital explorations.

Visualization during exploration of the orbit is hampered by herniation of orbital fat around the malleable retractor in posterior orbital dissection. A thin stiff disc of plastic sheeting, placed between the dissected orbital soft tissue contents and the malleable retractor, reduces prolapse of fat around the edges of the orbital retractor. The technique is utilized in acute fracture treatment and in late orbital reconstruction. The malleable retractor, placed below the plastic disc, may be easily taken in and out of the orbit with reduced damage of fat and enhanced visualization. Retractor damage to fat is reduced and visualization enhanced. The plastic sheet may be inserted over an intact posterior bony "ledge" in floor fractures, allowing accurate placement of bone grafts under direct visualization. Additionally, the use of a segment of flexible rubber sheeting sewed over a subciliary incision margin reduces traction injury, drill and cautery damage to eyelid skin.

Changes in area and thickness of expanded and unexpanded axial pattern skin flaps in pigs.

The pig buttock flap model was used firstly, to compare changes in expanded axial skin flap area with control flaps prior to flap elevation and 3 or 4 months post-flap inset and, secondly, to compare the thickness of expanded and control flaps at elevation and for 3 to 6 months post-flap inset. Following 5 weeks' expansion and prior to elevation, the expanded tissue had gained a significant 63.3% mean increase in area compared with the control tissue (p less than 0.01). Immediately post elevation and inset, the expanded flaps were still significantly larger than the control flaps by a mean 29.8% (p less than 0.01) but had lost 56% of the original area gained. Little change in area occurred in the 3 months post-flap inset as the expanded flaps were still a mean 23.4% larger than the control (p less than 0.01). Dermal and cellular non-keratinised epidermal layers thickened markedly in expanded skin compared to control skin. Following elevation and inset of the flaps, both dermis and epidermis thickened in expanded and control flaps.

Comparative viability of expanded and unexpanded axial pattern skin flaps in pigs.

A comparison of viable areas of axial pattern flaps post inset was made between expanded and non-expanded pig buttock island flaps. The deep circumflex iliac artery and vein supply approximately the proximal 14 x 10 cm area of this flap. Larger flaps were raised on expanded and control sides of eight pigs to determine if expansion increased the area of survival. In six of eight pigs whose initial tissue expansion did not create more than approximately a 50% increase in skin area, the expanded flaps had a statistically significant increase in viable skin area (proportionally) 10 days post inset than their control flaps (p less than 0.05). Two other pigs did not conform to this pattern. Their initial tissue expansion was greater than 50%, and the resulting area of flap viability was proportionally less in expanded flaps than the control flaps.

The reverse latissimus dorsi muscle flap for closure of meningomyelocele.

Closure of the meningomyelocele wound requires stable coverage of the dural repair. In the case presented, multiple conventional attempts at reconstruction failed. A modification of the "reverse" latissimus dorsi flap is presented that successfully managed this low lumbar defect.

Osteoradionecrosis of the olecranon: treatment by radial forearm flap.

Osteoradionecrosis of the olecranon is an unusual pathologic entity, treated best by debridement and wound closure using vascularized tissue. Local skin is often unavailable for flap design and transposition. The radial forearm flap can be isolated on a proximal vascular pedicle and transposed to cover the wound. In the case presented, healing was brisk and complete, allowing early elbow mobilization. Although the donor site is not easily concealed, no functional impairment results from flap elevation and all full-thickness wounds are confined to the involved extremity.

Protein metabolism and fibrosis in experimental canine obstructive lymphedema.

Obstructive lymphedema is a pathologic condition resulting in the accumulation and stagnation of serum proteins in the lymphatics and interstitial spaces. In a canine model of obstructive lymphedema, one limb was rendered lymphedematous, and various biochemical parameters were determined in this and an unaffected control limb. Both lymph and interstitial fluid had significantly decreased acid proteinase activity (comprising mostly cathepsin D-like enzymes) and neutral proteinase activity (comprising metallo, sulfhydryl, and serine proteinases, and collagenase). Possible reasons for these decreases could be: (1) saturation of macrophages and their surrounding environment with whole or partially digested proteins, or (2) elevation in the levels of circulating inhibitors like alpha 2-macroglobulin. The lymphedematous skin was significantly thicker than control skin and had elevated levels of collagen. However, unlike some fibrotic conditions, the relative proportions of types I, III, and V collagen, as determined by pepsin solubilization and neutral salt fractionation of the collagen fibrils, were similar to those found in normal skin. It is speculated that a decrease in the breakdown of collagen by collagenase and a continuing synthesis of collagen by fibroblasts led to an imbalance in favor of collagen deposition in the skin.

The free radial forearm flap with and without bone for closure of large palatal fistulae.

Three cases of palatal fistulae closed by microvascular transfer of radial forearm flaps are presented. Vascularised bone was included in one flap and all operations were free of complications. Facial scarring is minimal and the secondary deformity in the arm has not been significant. It is suggested that this procedure presents fewer difficulties than other techniques for treating the large palatal fistula and may prevent the collapse of the alveolar arch which follows the scarring associated with closure by local tissue.

The gluteus maximus musculocutaneous island flap: refinements in design and application.

The gluteus maximus island musculocutaneous flap has been described using a variety of designs. We employ an island whose long axis is directed toward the pressure sore, minimizing tension in wound closure. Skin overlying the greater trochanter is avoided. Previously undermined skin can be included in the flap. Fifty patients with ischial or sacral pressure sores have been managed by this technique. Superficial dehiscence occurred in 13 percent of patients, and deep dehiscence occurred in 10 percent. The dehiscence closed spontaneously in all but one patient. Forty-nine of the 50 patients experienced complete wound healing at the pressure sore site. The patients have been observed for an average of 20 months (range 3 to 38 months), with one recurrent pressure sore seen at 28 months postoperatively. The gluteus maximus musculocutaneous island flap has proven to be both reliable in healing and durable over the observed interval.