Definition of Critical Periods for Hedgehog Pathway Antagonist-Induced Holoprosencephaly,Cleft Lip,and Cleft Palate

The Hedgehog (Hh) signaling pathway mediates multiple spatiotemporally-specific aspects of brain and face development. Genetic and chemical disruptions of the pathway are known to result in an array of structural malformations, including holoprosencephaly (HPE), clefts of the lip with or without cleft palate (CL/P), and clefts of the secondary palate only (CPO). Here, we examined patterns of dysmorphology caused by acute, stage-specific Hh signaling inhibition. Timed-pregnant wildtype C57BL/6J mice were administered a single dose of the potent pathway antagonist vismodegib at discrete time points between gestational day (GD) 7.0 and 10.0, an interval approximately corresponding to the 15^th to 24^th days of human gestation. The resultant pattern of facial and brain dysmorphology was dependent upon stage of exposure. Insult between GD7.0 and GD8.25 resulted in HPE, with peak incidence following exposure at GD7.5. Unilateral clefts of the lip extending into the primary palate were also observed, with peak incidence following exposure at GD8.875. Insult between GD9.0 and GD10.0 resulted in CPO and forelimb abnormalities. We have previously demonstrated that Hh antagonist-induced cleft lip results from deficiency of the medial nasal process and show here that CPO is associated with reduced growth of the maxillary-derived palatal shelves. By defining the critical periods for the induction of HPE, CL/P, and CPO with fine temporal resolution, these results provide a mechanism by which Hh pathway disruption can result in “non-syndromic” orofacial clefting, or HPE with or without co-occurring clefts. This study also establishes a novel and tractable mouse model of human craniofacial malformations using a single dose of a commercially available and pathway-specific drug.     

Asymmetric limb malformations in a new transgene insertional mutant,footless

Six to eight copies of a transgene integrated into mouse chromosome 15 resulting in a new transgene insertional mutant, Footless, presenting with malformations of the limbs, kidney, and soft palate. Homozygotes possess a unique asymmetric pattern of limb truncations. Posterior structures from the autopod and zeugopod of the hindlimbs are missing with left usually more severely affected than right. In contrast, anterior structures are missing from the right forelimbs. The left forelimb is usually normal except for the absence of the distal telephalanges and nails. These structures are absent on all formed digits. In situ hybridization assays examined the expression of Shh, dHand, Msx2, Fgf8, En1, and Lmx1b in mutant limb buds and indicated normal establishment of the anterior/posterior and dorsal/ventral axes of the developing limbs. However, dysmorphology of the apical ectodermal ridge was observed in the mutant limb buds.     

Epidemiological and genetic study of 200 cases of oral cleft in the Emilia Romagna region of northern Italy

Epidemiological and genetic variables in clefts were analyzed during the years 1978-1986 in a case-control study of congenital malformations in the Emilia Romagna region of northern Italy. Among 150,168 newborns, 200 cases of cleft were detected, yielding a prevalence of 1.33 per 1,000. These clefts consisted of 112 (0.075%) cases of cleft lip with or without cleft palate (CL +/- P) and 88 cases (0.058%) of cleft palate (CP). Coexisting abnormalities were found in 32% of cases. The heritability coefficient of CL +/- P was 0.84. No cluster in time or space could be demonstrated. Epilepsy was the only maternal risk factor found to be correlated with clefts. A predominance of males was found among CL +/- P cases.     

Distal 4p microdeletion in a case of Wolf-Hirschhorn syndrome with congenital diaphragmatic hernia

BACKGROUND: Wolf-Hirschhorn syndrome (WHS) is a well-known genetic condition characterized by typical facial anomalies, midline defects, skeletal anomalies, prenatal and postnatal growth retardation, hypotonia, mental retardation, and seizures. Affected patients with a microdeletion on distal 4p present a milder phenotype that lacks congenital malformations. WHS is rarely associated with congenital diaphragmatic hernia (CDH), and only 8 cases are reported in the literature. In almost all cases of CDH and WHS a large deletion of the short arm of chromosome 4 is present. CASE: A microdeletion of 2.6 Mb on distal 4p associated with CDH and multiple congenital malformations (i.e., cleft palate) is reported for the first time. CONCLUSIONS: Such a microdeletion should prompt a molecular study for WHS when in a fetus/newborn with CDH the association with cleft lip/palate and typical facial appearance (flat facial profile, hypertelorism) is found.     

Periconceptional multivitamin intake during early pregnancy, genetic variation of acetyl-N-transferase 1 (NAT1), and risk for orofacial clefts

BACKGROUND: Periconceptional supplementation of multivitamins that include folic acid have been shown to prevent several birth defects, including neural tube defects and orofacial clefts. We investigated whether polymorphic variants of fetal acetyl-N-transferase 1 (NAT1), an enzyme involved in the catabolism of folates, differentially interacted with maternal multivitamin use during early pregnancy to alter the risk of delivering an infant with an orofacial cleft malformation. METHODS: Using a large population-based case-control study, we genotyped 421 California infants born with an isolated cleft and 299 controls for two NAT1 polymorphisms. RESULTS: Compared to the homozygous wild-type genotypes, odds ratios for isolated cleft lip with/without cleft palate were slightly increased among infants who were homozygous for the variant alleles of NAT1 1088 and 1095. For isolated cleft palate, no similar associations with these two NAT1 variants were observed. For NAT1 1088 genotypes, we did not observe any differential risks for clefts related to maternal multivitamin intake. For NAT1 1095 genotypes, however, we found a two-fold higher risk for isolated cleft lip with/without cleft palate among infants who were homozygous for the variant allele and whose mothers did not take multivitamins during early pregnancy. CONCLUSIONS: We found evidence suggestive of an interaction between the NAT1 1095 polymorphism and lack of maternal multivitamin use that increased risks of isolated cleft lip with/without cleft palate.     

Orofacial clefts and infections during pregnancy

Orofacial clefts (OFCs) are common congenital malformations of the lip, palate, or both caused by complex genetic and environmental factors. Specific antibodies against viruses influenza, rubella, cytomegalovirus, Epstein-Barr, parotitis and hepatitis B were investigated serologically in children with orofacial clefts and in their mothers. The results were compared with those obtained in control children and their mothers. Evaluation of the results and their statistical processing supports the assumption that infection during pregnancy may have occurred in the series studied induced by viruses of influenza, rubella, cytomegalovirus and possibly also by the Epstein-Barr virus. No association with the viruses of hepatitis B and parotitis was established.     

Cleft lip with and without cleft palate in blacks: an analysis of 81 patients

The clinical records of 81 black patients with cleft lip with or without cleft palate were reviewed. Four had midline clefts. Of the remaining 77, 45 were unilateral (left 28, right 17), with 11 of these involving only the primary palate. Bilateral clefts were seen in 32, with only 2 involving just the primary palate. Males and females were approximately equal in number. Two were associated with EEC syndrome. Other congenital anomalies were seen in 9 patients. The family history was positive for clefts in 5 of 65 patients (7.7 percent). A review of 255 white patients with cleft lip with or without cleft palate revealed a positive family history in 94 (37 percent). The difference was statistically significant.     

Genetic approaches to identify disease genes for birth defects with cleft lip/palate as a model

BACKGROUND: Understanding the etiology of birth defects is an important step toward developing improved treatment and preventive strategies. Most birth defects have an underlying genetic basis, ranging from single genes playing dominant or recessive roles in Mendelian disorders to a mixture of contributions from multiple genes and environmental triggers in complex traits. The purpose of this article is to provide an overview of genetic approaches to identifying disease genes for genetically complex birth defects. METHODS: A review of the literature describing successes and limitations for identifying disease genes for complex traits was conducted. RESULTS: Cleft lip and cleft palate are common congenital anomalies with significant medical, psychological, social, and economic ramifications. The Online Mendelian Inheritance in Man catalog (OMIM; http://www3.ncbi.nlm.nih.gov/Omim) lists more than 400 single-gene causes of clefts of the lip and/or palate. Genetic causes of clefting also include chromosomal rearrangements, genetic susceptibility to teratogenic exposures, and complex genetic contributions of multiple genes. CONCLUSIONS: Genetic causes of birth defects can be identified using an increasingly powerful combination of careful sample collection, molecular analytic methods, and statistical evaluations. We will describe a range of approaches to search for genetic factors of birth defects and use our own work with cleft lip and palate as a model.     

Facial clefts in Saudi Arabia: an epidemiologic analysis in 179 patients

One hundred and seventy-nine consecutive cases of facial clefts that were treated at the King Khalid University Hospital, in Riyadh, Saudi Arabia, were analyzed for an epidemiologic study. Isolated cleft lip was present in 38 percent, cleft of lip and palate in 37.4 percent, and cleft of the posterior palate in only 22.4 percent. There was a male preponderance in all types. In cases of cleft lip with or without cleft palate, the more commonly affected side was the left, followed by bilateral cases. Associated malformations were present in 13.4 percent. A positive family history was found in 26.8 percent of cases. A significant number of patients (7.8 percent) were first seen at more than 10 years of age. The incidence of facial clefts at this hospital was 0.3 per 1000 live births, computed over a period of 6 years. This incidence is significantly lower than that reported from European and Far Eastern countries.     

Congenital malformations associated with anencephaly in liveborn and stillborn infants

Records from the population-based British Columbia Health Surveillance Registry were examined, and a total of 456 infants with anencephaly (181 liveborns, 275 stillborns) were identified. Registry records list up to four congenital malformations per individual, and the records of the study cohort were reviewed for the presence of additional malformations. A total of 12.7% of infants (14.4% liveborns, 11.6% stillborns) had congenital malformations in addition to anencephaly. The frequencies of specific congenital malformations (e.g., talipes, cleft lip and/or palate, omphalocele) in infants with anencephaly were compared with the frequencies of these malformations in the general population of liveborns. In addition, the types of additional congenital malformations in liveborn anencephalics compared to stillborns were looked at. The similarity suggests that it is not just the presence of these additional congenital malformations that leads to death in utero. The data provide further evidence for etiological heterogeneity in neural tube defects.     

General body growth in children with cleft palate and related disorders: age differences

Clefts of the lip and palate, separately or in combination, are among the most frequent congenital defects seen today. Their etiology is heterogeneous and may include hormonal factors, which suggest the possibility of growth effects. Whether affected children are smaller than others has not been determined. We recently showed that growth status is associated with type of cleft. We hypothesized genetic alterations in metabolic pathways that alter prenatal growth, producing clefts; some of these alterations also alter postnatal growth. Since the levels of growth-regulating hormones change during ontogeny, we expected age differences in the degree of growth deficit seen. To test this hypothesis, we examine here the cross-sectional means and distributions of standard deviation (z) scores for height and body mass indices (BMIs) for 144 children with the diagnoses unilateral cleft lip and palate (uCLP) and isolated cleft palate (iCP). We find that alteration in growth status is associated with age group as well as sex and diagnosis.     

Rare facial clefts: treatment during charity missions in developing countries

During 10 charity missions in developing countries, 14 patients of a total of 374 children with cleft lip and palate deformities were treated for rare facial clefts. There were three midline clefts (Tessier no. 0 cleft, n = 1; Tessier no. 14 cleft, n = 2), four oblique facial clefts (Tessier no. 3 cleft, n = 2; Tessier no. 5 cleft, n = 2), and seven lateral facial clefts (Tessier no. 7 cleft). Surgical treatment focused on cleft repair by soft-tissue reconstruction apart from two Tessier no. 14 clefts, in which the bony gap was also closed using bone grafts from the iliac crest. The postoperative course was uneventful except for one local wound infection that was treated successfully using oral antibiotics. This article summarizes the authors' experience with the surgical management of these malformations and considers the limitations under conditions of charity missions in developing countries. Furthermore, some rare forms of cleft formation are added to the existing literature.     

FAF1, a gene that is disrupted in cleft palate and has conserved function in zebrafish

Cranial neural crest (CNC) is a multipotent migratory cell population that gives rise to most of the craniofacial bones. An intricate network mediates CNC formation, epithelial-mesenchymal transition, migration along distinct paths, and differentiation. Errors in these processes lead to craniofacial abnormalities, including cleft lip and palate. Clefts are the most common congenital craniofacial defects. Patients have complications with feeding, speech, hearing, and dental and psychological development. Affected by both genetic predisposition and environmental factors, the complex etiology of clefts remains largely unknown. Here we show that Fas-associated factor-1 (FAF1) is disrupted and that its expression is decreased in a Pierre Robin family with an inherited translocation. Furthermore, the locus is strongly associated with cleft palate and shows an increased relative risk. Expression studies show that faf1 is highly expressed in zebrafish cartilages during embryogenesis. Knockdown of zebrafish faf1 leads to pharyngeal cartilage defects and jaw abnormality as a result of a failure of CNC to differentiate into and express cartilage-specific markers, such as sox9a and col2a1. Administration of faf1 mRNA rescues this phenotype. Our findings therefore identify FAF1 as a regulator of CNC differentiation and show that it predisposes humans to cleft palate and is necessary for lower jaw development in zebrafish.     

Median facial clefts in Xenopus laevis: roles of retinoic acid signaling and homeobox genes

The upper lip and primary palate form an essential separation between the brain, nasal structures and the oral cavity. Surprisingly little is known about the development of these structures, despite the fact that abnormalities can result in various forms of orofacial clefts. We have uncovered that retinoic acid is a critical regulator of upper lip and primary palate development in Xenopus laevis. Retinoic acid synthesis enzyme, RALDH2, and retinoic acid receptor gamma (RARγ) are expressed in complementary and partially overlapping regions of the orofacial prominences that fate mapping revealed contribute to the upper lip and primary palate. Decreased RALDH2 and RARγ result in a median cleft in the upper lip and primary palate. To further understand how retinoic acid regulates upper lip and palate morphogenesis we searched for genes downregulated in response to RARγ inhibition in orofacial tissue, and uncovered homeobox genes lhx8 and msx2. These genes are both expressed in overlapping domains with RARγ, and together their loss of function also results in a median cleft in the upper lip and primary palate. Inhibition of RARγ and decreased Lhx8/Msx2 function result in decreased cell proliferation and failure of dorsal anterior cartilages to form. These results suggest a model whereby retinoic acid signaling regulates Lhx8 and Msx2, which together direct the tissue growth and differentiation necessary for the upper lip and primary palate morphogenesis. This work has the potential to better understand the complex nature of the upper lip and primary palate development which will lead to important insights into the etiology of human orofacial clefts.     

R-spondin2 expression in the apical ectodermal ridge is essential for outgrowth and patterning in mouse limb development

Mouse R-spondin2 (Rspo2) is a member of the R-spondin protein family, which is characterized by furin-like cysteine-rich domains and a thrombospondin type 1 repeat. R-spondin is a secreted molecule that activates Wnt/ β -catenin signaling. Rspo2 -deficient mice were generated to investigate the function of mouse Rspo2 during embryonic development. The homozygous mutant forelimb showed defects in distal phalanges and nail structures, and the digits were anomalous in shape. The homozygous mutant hindlimb showed more severe malformations, including lack of digits and zeugopod components. Rspo2 is expressed in the apical ectodermal ridge (AER) of the developing limb. Fgf8 expression in the AER was significantly lower in the homozygous mutant forelimb than in the wild-type forelimb and it was disturbed along the dorsoventral axis. In the homozygous mutant hindlimb, Fgf8 and Fgf4 expression in the posterior AER and Sonic hedgehog expression in the zone of polarizing activity (ZPA) were reduced. The homozygous mutant hindlimb also showed expansion of Wnt7a expression in the dorsal ectoderm toward the ventral side. This study shows that Rspo2 is critical for maintenance of the AER and for growth and patterning in limb development.     

Oral-Facial Clefts and Associated Malformations in the Squirrel Monkey (Saimiri sciureus)

Of the 414 squirrel monkey pregnancies recorded at this institution since 1977, seven (1.7%) have resulted in offspring with clefts of the lip and/or palate. Associated malformations include a ventricular septal defect, renal agenesis, anal atresia, axial skeletal anomalies, and craniorachischisis (anencephaly and spina bifida). Three of these infants are the result of consanguineous matings.     

Familial risk of oral clefts by morphological type and severity: population based cohort study of first degree relatives

Objective To estimate the relative risk of recurrence of oral cleft in first degree relatives in relation to cleft morphology.Design Population based cohort study.Setting Data from the medical birth registry of Norway linked with clinical data on virtually all cleft patients treated in Norway over a 35 year period.Participants 2.1 million children born in Norway between 1967 and 2001, 4138 of whom were treated for an oral cleft.Main outcome measure Relative risk of recurrence of isolated clefts from parent to child and between full siblings, for anatomic subgroups of clefts.Results Among first degree relatives, the relative risk of recurrence of cleft was 32 (95% confidence interval 24.6 to 40.3) for any cleft lip and 56 (37.2 to 84.8) for cleft palate only (P difference=0.02). The risk of clefts among children of affected mothers and affected fathers was similar. Risks of recurrence were also similar for parent-offspring and sibling-sibling pairs. The “crossover” risk between any cleft lip and cleft palate only was 3.0 (1.3 to 6.7). The severity of the primary case was unrelated to the risk of recurrence.Conclusions The stronger family recurrence of cleft palate only suggests a larger genetic component for cleft palate only than for any cleft lip. The weaker risk of crossover between the two types of cleft indicates relatively distinct causes. The similarity of mother-offspring, father-offspring, and sibling-sibling risks is consistent with genetic risk that works chiefly through fetal genes. Anatomical severity does not affect the recurrence risk in first degree relatives, which argues against a multifactorial threshold model of causation.     

Experimental studies on a lethal gene (t) in the Mexican axolotl, Ambystoma mexicanum

In the Mexican axolotl, Ambystoma mexicanum, the developmental mutation lethal t is inherited as a simple Mendelian recessive. Mutant larvae failed to feed and died, on the average, 17 days after hatching. Unfed wild-type larvae died an average of 23 days after hatching. By 15 days, forelimb development had progressed further in the wild type; a cartilaginous scapula and humerus were present, but no cartilage was seen in the mutant limb. Histological examination indicated that the visceral cartilage may also be abnormal, and the rectus cervicus muscle was found to have fewer and smaller fibers. Though the mutant was not rescued by parabiosis with wild-type embryos, transplants of presumptive gill and limb tissue to wild-type hosts survived, indicating that the mutation is not an autonomous cell lethal.     

唇腭裂患儿下调表达的miRNA相关研究

目的:唇腭裂是口腔颌面部最常见的先天性畸形,危害严重。mi RNA在细胞分化,生物发育及疾病发生发展过程中发挥巨大作用,越来越多的受到科研人员的关注。本课题对唇腭裂患儿下调表达的mi RNA与唇腭裂相关性进行验证研究,为mi RNA应用在唇腭裂防治中奠定一定的基础。方法:通过芯片分析方法检测唇腭裂患儿表达下调的mi RNA,利用MIRDB、TARGETSCAN-VERT和RNA22-HSA这三个生物信息学软件对唇腭裂患儿表达下调的mi RNA进行靶基因预测分析,验证候选mi RNA与唇腭裂具有相关性。结果:得出唇腭裂患儿中出现差异表达下调的mi RNA有hsa-mi R-3119,hsa-mi R-3915等73个,其中hsa-mi R-3611对应的靶基因GABRB3和hsa-mi R-764对应的靶基因F13A1有文献报道与唇腭裂具有相关性。结论:hsa-mi R-3611,hsa-mi R-764可能与唇腭裂的发生存在关联。