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.     

Distinct functions for Bmp signaling in lip and palate fusion in mice

Previous work suggested that cleft lip with or without cleft palate (CL/P) is genetically distinct from isolated cleft secondary palate (CP). Mutations in the Bmp target gene Msx1 in families with both forms of orofacial clefting has implicated Bmp signaling in both pathways. To dissect the function of Bmp signaling in orofacial clefting, we conditionally inactivated the type 1 Bmp receptor Bmpr1a in the facial primordia, using the Nestin cre transgenic line. Nestin cre; Bmpr1a mutants had completely penetrant, bilateral CL/P with arrested tooth formation. The cleft secondary palate of Nestin cre; Bmpr1a mutant embryos was associated with diminished cell proliferation in maxillary process mesenchyme and defective anterior posterior patterning. By contrast, we observed elevated apoptosis in the fusing region of the Nestin cre; Bmpr1a mutant medial nasal process. Moreover, conditional inactivation of the Bmp4 gene using the Nestin cre transgenic line resulted in isolated cleft lip. Our data uncover a Bmp4-Bmpr1a genetic pathway that functions in lip fusion, and reveal that Bmp signaling has distinct roles in lip and palate fusion.     

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.     

Corticosteroid use during pregnancy and risk of orofacial clefts

Background

The association between the risk of orofacial clefts in infants and the use of corticosteroids during pregnancy is unclear from the available evidence. We conducted a nationwide cohort study of all live births in Denmark over a 12-year period.

Methods

We collected data on all live births in Denmark from Jan. 1, 1996, to Sept. 30, 2008. We included live births for which information was available from nationwide health registries on the use of corticosteroids during pregnancy, the diagnosis of an orofacial cleft and possible confounders.

Results

There were 832 636 live births during the study period. Exposure to corticosteroids during the first trimester occurred in 51 973 of the pregnancies. A total of 1232 isolated orofacial clefts (i.e., cleft lip, cleft palate, or cleft lip and cleft palate) were diagnosed within the first year of life, including 84 instances in which the infant had been exposed to corticosteroids during the first trimester of pregnancy. We did not identify any statistically significant increased risk of orofacial clefts associated with the use of corticosteroids: cleft lip with or without cleft palate, prevalence odds ratio (OR) 1.05 (95% confidence interval [CI] 0.80–1.38]; cleft palate alone, prevalence OR 1.23 (95% CI 0.83–1.82). Odds ratios for risk of orofacial clefts by method of delivery (i.e., oral, inhalant, nasal spray, or dermatologic and other topicals) were consistent with the overall results of the study and did not display significant heterogeneity, although the OR for cleft lip with or without cleft palate associated with the use of dermatologic corticosteroids was 1.45 (95% CI 1.03–2.05).

Interpretation

Our results add to the safety information on a class of drugs commonly used during pregnancy. Our study did not show an increased risk of orofacial clefts with the use of corticosteroids during pregnancy. Indepth investigation of the pattern of association between orofacial clefts and the use of dermatologic corticosteroids during pregnancy indicated that this result did not signify a causal connection and likely arose from multiple statistical comparisons.The anti-inflammatory and immuno-suppressive properties of corticosteroids in pharmacotherapeutic doses has a wide range of clinical uses, such as for the treatment of asthma, atopic dermatitis and other allergic conditions, autoimmune diseases and cancer. However, caution is warranted for the use of corticosteroid medications during pregnancy. Corticosteroid use during pregnancy has been associated with orofacial clefts in animals, and similar risks in humans are suspected.^1,2 The available epidemiologic evidence favours an association, but many of the studies that have been done have been limited by recall bias and a lack of statistical power. The association between risk of orofacial clefts and the use of corticosteroids during pregnancy remains unclear.^3–10We conducted a nationwide cohort study in Denmark with independent and prospective determination of corticosteroid use during pregnancy and the diagnosis of orofacial clefts. Our study comprised all live births from January 1996 to September 2008.     

Birth prevalence of cleft lip and palate in British Columbia between 1952 and 1986: stability of rates.

We examined the birth prevalence of cleft lip with or without cleft palate and of isolated cleft palate in British Columbia between 1952 and 1986 using the data of the BC Health Surveillance Registry. The rates fluctuated over the study period, but linear trend analysis showed no increase or decrease for cleft lip with or without cleft palate; however, there was a significant increase for isolated cleft palate, attributed to improved ascertainment around 1963-66. Given the possible effects of newer agents used in both silviculture and agriculture, as well as the general concern over drugs and other environmental agents, such a long-term monitoring program is important. Furthermore, if significant clustering occurs, good background data are essential for comparison. The general public''s perception is that the rates of birth defects are increasing. Our findings should give some reassurance with respect to orofacial clefts.     

Maternal multivitamin use and orofacial clefts in offspring

BACKGROUND: Cleft lip with or without cleft palate (CLP) and cleft palate alone (CP) affect approximately 1 in 1000 infants and 1 in 2,500 infants, respectively. Studies of the relation between orofacial clefts and multivitamins or folic acid have been inconsistent. METHODS: We used data from a population-based case-control study involving 309 nonsyndromic cleft-affected births (222 with CLP, 87 with CP) and 3,029 control births from 1968 to 1980 to evaluate the relation between regular multivitamin use and the birth prevalence of orofacial clefts. RESULTS: We found a 48% risk reduction for CLP (odds ratio = 0.52, 95% confidence interval = 0.34-0.80) among mothers who used multivitamins during the periconceptional period or who started multivitamin use during the first postconceptional month, after controlling for several covariates. The risk reduction for CP was less than those for CLP (odds ratio = 0.81, 95% confidence interval = 0.44-1.52); however, a small number of CP cases limited interpretation. No risk reductions for CLP or CP were found for women who began multivitamin use in the second or third month after conception. CONCLUSIONS: The magnitude of the risk reduction in our study is comparable to those of other recent studies; our study does not support the contention that only large dosages of folic acid are needed to prevent orofacial clefts. More studies are needed to test the effects of multivitamins and varying dosages of folic acid on the recurrence and/or occurrence of orofacial clefts to provide information needed to determine possible prevention strategies. Published 2001 Wiley-Liss, Inc.     

Socioeconomic measures,orofacial clefts,and conotruncal heart defects in California

OBJECTIVE : To examine the association of multiple measures of socioeconomic status (SES) with risks of orofacial clefts and conotruncal heart defects. DESIGN : Data were from a recent population‐based case‐control study conducted in California that included 608 patients with orofacial clefts, 277 patients with conotruncal heart defects, and 617 nonmalformed controls. RESULTS : The odds ratio for the worst versus best score on a household‐level SES index was strongest for cleft lip with or without palate, at 1.7 (95% confidence interval, 0.9–3.4); the odds ratios for this comparison were closer to 1 and less precise for the other defect groups. An index based on neighborhood‐level SES was also not associated with increased risk of the studied defects. CONCLUSIONS : This detailed analysis of SES and selected birth defects did not suggest worse SES was associated with increased risk of the studied defects, with the possible exception of cleft lip with or without cleft palate. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Family history and socioeconomic risk factors for non-syndromic cleft lip and palate: A matched case-control study in a less developed country

Introduction. From an epidemiological point of view, non-syndromic orofacial clefts are the most common oral congenital deformities worldwide. Objective. Family histories were traced and socioeconomic risk factors were identified for non-syndromic cleft lip with or without cleft palate. Material and methods. A case-control study was carried out with 208 cases of non-syndromic cleft lip with or without cleft palate, and matched by age and sex with 416 controls. Cases were patients attending a referral clinic from 2002 through 2004 in Campeche, Mexico. A questionnaire was administered to collect sociodemographic and socioeconomic variables as well as familial background relevant to non-syndromic cleft lip with or without cleft palate. Conditional logistic regression models were used; adjusted odds ratios and 95% confidence intervals were calculated. Results. In the multivariate model, the following risk factors were identified: 1) low socioeconomic status; 2) birth in the southern region of Campeche state; 3) home delivery or delivery in a publicly funded hospital; 4) occurrence of prior non-syndromic cleft lip with or without cleft palate cases in the father′s or mother′s family: 5) having a sibling with non-syndromic cleft lip with or without cleft palate; 6) the proband having another malformation, and 7) a history of infections during pregnancy. Prenatal care consisting of vitamin supplementation was a protective factor for non-syndromic cleft lip with or without cleft palate (odds ratio=0.29). Conclusions. A "social gradient in health" was seen to link oral malformation with diet components, and several socioeconomic and socio-demographic factors broadly encompassed in low socioeconomic status. Further characterization of risk factors will guide the assemblage of a pro-active counseling and prevention program for families at risk for non-syndromic cleft lip and cleft palate.     

Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and nonsyndromic orofacial clefts susceptibility in a southern Chinese population

Nonsyndromic orofacial clefts (NSOC) are one of the most common congenital anomalies in humans. Great efforts have been taken to unravel its genetic background. Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in folate metabolism and two of its functional polymorphisms, MTHFR C677T and MTHFR A1298C, might be associated with NSOC susceptibility. The aim of the present study was to investigate their associations with risks of NSOC in a southern Chinese population. We found that MTHFR 677 TT and 677 CT/TT were associated with increased risk of cleft lip with or without cleft palate; meanwhile, MTHFR 1298 AC and 1298 AC/CC had protective effects against cleft lip with or without cleft palate. In further stratified analysis, we found that MTHFR 677 CT contributed to elevated risk of cleft lip only, as did MTHFR 677 CT/TT. On the contrary, MTHFR 1298 AC and 1298 AC/CC appeared to be protective against cleft lip with cleft palate. These results suggested that these two polymorphisms were involved in the development of NSOC in a southern Han Chinese population.     

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.     

Sequential programs of retinoic acid synthesis in the myocardial and epicardial layers of the developing avian heart

Endogenous patterns of retinoic acid (RA) signaling in avian cardiac morphogenesis were characterized by localized expression of a key RA-synthetic enzyme, RALDH2, which displayed a biphasic pattern during heart development. RALDH2 immunoreactivity was initially apparent posterior to Hensen's node of stage 5-6 embryos and subsequently in somites and unsegmented paraxial and lateral plate mesoderm overlapping atrial precursors in the cardiogenic plate of stage 9- embryos. Initial RALDH2 synthesis in the posterior myocardium coincided with activation of the AMHC1 gene, a RA-responsive marker of inflow heart segments. A wave of RALDH2 synthesis then swept the myocardium in a posterior-to-anterior direction, reaching the outflow tract by stage 13, then fading from the myocardial layer. The second phase of RALDH2 expression, initiated at stage 18 in the proepicardial organ, persisted in migratory epicardial cells that completely enveloped the heart by stage 24. Early restriction of RALDH2 expression to the posterior cardiogenic plate, overlapping RA-inducible gene activation, provides evidence for commitment of posterior avian heart segments by localized production of RA, whereas subsequent RALDH2 expression exclusively in the migratory epicardium suggests a role for the morphogen in ventricular expansion and morphogenesis of underlying myocardial tissues.     

视黄酸核受体γ重组腺病毒构建及其鉴定

目的构建携带大鼠视黄酸核受体γ（retinoic acid receptor γ,RARγ）的重组腺病毒,为研究RAR3,在骨髓间充质干细胞（mesenchymal stem cells,MSCs）成神经分化中的作用奠定基础。方法体外扩增大鼠础研基因,将其定向克隆至腺病毒穿梭质粒pAd Trace—TOX构建重组质粒pAdTrace—RARγ,并在BJ5183菌中与骨架质粒pAd Easy-1重组获得腺病毒载体pad—RARγ,Pac I酶切后转染HEK293细胞包装腺病毒。腺病毒Ad—RARγ感染大鼠MSCs,Real—time PCR和Western印迹检测RARγ的表达。结果PCR,酶切及测序均证实础研正确克隆至腺病毒质粒载体中,Ad—RARγ对MSCs感染率达60％～70％,并明显增强础研基因和蛋白的表达。结论成功构建携带RARγ的重组腺病毒,并具有上调大鼠MSCsRA脚基因和蛋白表达的功能。     

Microarray analysis of murine palatogenesis: temporal expression of genes during normal palate development

The mammalian face is assembled in utero in a series of complex and interdependent molecular, cell and tissue processes. The orofacial complex appears to be exquisitely sensitive to genetic and environmental influence and this explains why clefts of the lip and palate are the most common congenital anomaly in humans (one in 700 live births). In this study, microarray technology was used to identify genes that may play pivotal roles in normal murine palatogenesis. mRNA was isolated from murine embryonic palatal shelves oriented vertically (before elevation), horizontally (following elevation, before contact), and following fusion. Changes in gene expression between the three different stages were analyzed with GeneChip microarrays. A number of genes were upregulated or downregulated, and large changes were seen in the expression of loricrin, glutamate decarboxylase, gamma-amino butyric acid type A receptor beta3 subunit, frizzled, Wnt-5a, metallothionein, annexin VIII, LIM proteins, Sox1, plakophilin1, cathepsin K and creatine kinase. In this paper, the changes in genetic profile of the developing murine palate are presented, and the possible role individual genes/proteins may play during normal palate development are discussed. Candidate genes with a putative role in cleft palate are also highlighted.     

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.     

Morphology of the orbital region in adults following the cleft lip-palate repair in childhood

Four measurements and two qualitative signs related to the orbits of 145 adult Caucasian cleft lip/palate patients operated on in childhood were compared with similar data on 100 normal Caucasian Canadians. The average interorbital distance in male patients with unilateral and bilateral cleft lip/palate was greater than in controls, while the interorbital distance in both male and female patients with isolated cleft palate was the same as that in controls. A hypertelorism increased interorbital distance of greater than 2 S.D. above the normal was recorded in 10 cleft patients out of 145, the maximum in male cleft patients being 48 mm and in female cleft patients 38 mm. Orbital eye fissure length asymmetry was seen only in the cleft study group while a dislocation of the eye fissure levels in the frontal plane was found both in patients with clefts and in controls. No direct relationship was found between the extent of the cleft and the incidence of hypertelorism, nor between the site of the cleft and eye fissure asymmetry in unilateral cleft lip/palate patients. The epicanthic fold was significantly more frequent in cleft lip/palate patients (28/145) than in controls (10/100). Anti-mongoloid eye fissure type was recorded only in patients with cleft but mongoloid eye fissure was present both in patients with clefts and controls.     

Medical sequencing of candidate genes for nonsyndromic cleft lip and palate

Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father). The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Étude du Polymorphisme Humain (CEPH) diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations.