Fine-mapping of 5q12.1-13.3 unveils new genetic contributors to caries.

Caries is a multifactorial disease and little is still known about the host genetic factors influencing susceptibility. Our previous genome-wide linkage scan has identified the interval 5q12.1-5q13.3 as linked to low caries susceptibility in Filipino families. Here we fine-mapped this region in order to identify genetic contributors to caries susceptibility. Four hundred and seventy-seven subjects from 72 pedigrees with similar cultural and behavioral habits and limited access to dental care living in the Philippines were studied. DMFT scores and genotype data of 75 single-nucleotide polymorphisms were evaluated in the Filipino families with the Family-Based Association Test. For replication purposes, a total 1,467 independent subjects from five different populations were analyzed in a case-control format. In the Filipino cohort, statistically significant and borderline associations were found between low caries experience and four genes spanning 13 million base pairs (PART1, ZSWIM6, CCNB1, and BTF3). We were able to replicate these results in some of the populations studied. We detected PART1 and BTF3 expression in whole saliva, and the expression of BTF3 was associated with caries experience. Our results suggest BTF3 may have a functional role in protecting against caries.

Regional analysis on the occurrence of oral clefts in South America.

The aim of this work was to search for unequal birth prevalence rates (BPRs) of cleft lip +/- cleft palate (CL/P), and cleft palate only (CPO), among different geographic areas in South America, and to analyze phenotypic characteristics and associated risk factors in each identified cluster. Included were 5,128 CL/P cases, 1,745 CPO cases, and 3,712 controls (like-sexed, non-malformed liveborn infant, born immediately after a malformed one, in the same hospital), over 4,199,630 consecutive births. They were ascertained between 1967 and 2004, in 190 maternity hospitals of the ECLAMC (Estudio Colaborativo Latinoamericano de Malformaciones Congénitas) network, in 102 cities of all 10 South American countries. Non-predefined geographical areas with significantly unusual cleft BPRs were identified with Kulldorf and Nagarwalla's spatial scan statistic, employing number of cases and births, and exact location of each hospital. Expected values were cleft BPRs registered for the entire ECLAMC hospital network. Syndromic and non-syndromic clefts were considered for cluster analysis, and phenotypic characterization, while only non-syndromic for risk factor analysis. Seven clusters for CL/P, and four for CPO, with unusual BPRs were identified. CL/P cases in high BPR areas were more severe than elsewhere in the sample, similar to a previous ECLAMC report on microtia. For CL/P, high BPR clusters were associated with high altitude above sea level, Amerindian ancestry, and low socioeconomic strata; low BPR clusters showed association with African Black ancestry. Advanced maternal age, a recognized risk factor for CPO, was also associated with the only identified geographic cluster for CPO.

Genetic evidence for the role of loci at 19q13 in cleft lip and palate.

BACKGROUND: Clefts of the lip and palate are common birth defects, affecting approximately 1 in 700 births worldwide. The aetiology of clefting is complex, with multiple genetic and environmental influences. METHODS: Genotype based linkage disequilibrium analysis was conducted using the family based association test (FBAT) and the likelihood ratio test (LRT). We also carried out direct sequencing of the PVR and PVRL2 candidate genes based on their homology to PVRL1, a gene shown previously to cause Margarita Island clefting. Participants included 434 patients with cleft lip with or without cleft palate or cleft palate only and their mothers from eight countries in South America, 205 nuclear triads (father-mother-affected child) from Iowa, 541 nuclear triads from Denmark, and 100 patients with cleft lip and palate from the Philippines. RESULTS: An allelic variant in the PVR gene showed statistically significant association with both South American and Iowa populations (p = 0.0007 and p = 0.0009, respectively). Direct sequencing of PVR and PVRL2 yielded 26 variants, including two rare amino acid changes, one in each gene, which were not seen in controls. CONCLUSIONS: We found an association between a common variant in a gene at 19q and isolated clefting in two heterogeneous populations. However, it is unclear from our data if rare variants in PVR and PVRL2 are sufficient to cause clefting in isolation.

Complete sequencing shows a role for MSX1 in non-syndromic cleft lip and palate.

MSX1 has been proposed as a gene in which mutations may contribute to non-syndromic forms of cleft lip and/or cleft palate. Support for this comes from human linkage and linkage disequilibrium studies, chromosomal deletions resulting in haploinsufficiency, a large family with a stop codon mutation that includes clefting as a phenotype, and the Msx1 phenotype in a knockout mouse. This report describes a population based scan for mutations encompassing the sense and antisense transcribed sequence of MSX1 (two exons, one intron). We compare the completed genomic sequence of MSX1 to the mouse Msx1 sequence to identify non-coding homology regions, and sequence highly conserved elements. The samples studied were drawn from a panethnic collection including people of European, Asian, and native South American ancestry. The gene was sequenced in 917 people and potentially aetiological mutations were identified in 16. These included missense mutations in conserved amino acids and point mutations in conserved regions not identified in any of 500 controls sequenced. Five different missense mutations in seven unrelated subjects with clefting are described. Evolutionary sequence comparisons of all known Msx1 orthologues placed the amino acid substitutions in context. Four rare mutations were found in non-coding regions that are highly conserved and disrupt probable regulatory regions. In addition, a panel of 18 population specific polymorphic variants were identified that will be useful in future haplotype analyses of MSX1. MSX1 mutations are found in 2% of cases of clefting and should be considered for genetic counselling implications, particularly in those families in which autosomal dominant inheritance patterns or dental anomalies appear to be cosegregating with the clefting phenotype.

Achondrogenesis type II with polydactyly.

We report on a newborn male infant who presented the typical findings of achondrogenesis type II (Langer-Saldino), and who also showed postaxial polydactyly on both feet and bilateral microtia. Polydactyly is frequently part of the short-rib syndromes, but has not been reported in achondrogenesis. The hypothesis of polydactyly as part of a contiguous gene syndrome is discussed.

Thumb/hallux duplication and preaxial polydactyly type I.

It was recently shown that hand postaxial polydactyly differed from foot postaxial polydactyly. The aim of this work was to test whether thumb and hallux duplication also had different clinical and epidemiological characteristics, depending on limb involvement. We studied 920 newborn infants with first digit duplication, ascertained among 3,444,374 births by the Latin-American Collaborative Study of Congenital Malformations (ECLAMC), from 1967 to 1995. Since biphalangeal thumb duplication or hallux duplication can occur in families with triphalangeal thumb or polysyndactylous propositi, these groups were also analyzed. The 715 isolated (nonsyndromal) cases (prevalence 2.08 per 10,000) were subdivided into five groups: thumb duplication (N = 568; prevalence: 1.65/10,000); hallux duplication (N = 82; prevalence: 0.24); thumb and/or hallux duplication plus syndactyly (polysyndactyly) (N = 37; prevalence: 0.11); triphalangeal thumb (N = 24; prevalence: 0.07), and thumb duplication plus hallux duplication (N = 4; prevalence: 0.01). Both thumb and hallux duplication groups showed a significant excess of males, and right sidedness was also more frequent in both of them, though without statistical significance for hallux duplication. Thumb duplication was more often unilateral (94.7% versus hallux duplication of 81.5%), and its prevalence was higher in Bolivia (3.37/10,000) than in the other 10 Latin-American countries included (1.62/10,000). In a subseries of 405 preaxial polydactylies with matched controls, a logistic regression analysis showed that birth weight and gestational age had an effect on the calculated risk of having an infant with thumb duplication, while first trimester vaginal bleeding had only a borderline effect. None of the polydactyly groups showed abnormal values for twinning, perinatal mortality, ethnicity, maternal education, parental ages, parity, parental subfertility, or consanguinity. There were 70/405 familial cases. Their pedigrees were compatible with autosomal dominant inheritance with a 9% penetrance for thumb duplication and hallux duplication and a 70% penetrance for triphalangeal thumb and polysyndactyly. Inheritance of thumb duplication and probably the untested inheritance of hallux duplication were also compatible with a four-locus multiplicative model. The observed differences in laterality, geographical distribution, birth weight, gestational age, and first trimester vaginal bleeding between thumb duplication and hallux duplication groups suggested that apparent preaxial polydactyly type 1 is a causally heterogeneous group.

Epidemiology of neural tube defects in South America.

We present data from the Latin American Collaborative Study of Congenital Malformations (ECLAMC) on prevalence rates and etiologic factor associations in neural tube defects. Two series of data are analyzed: the A series, including 740,139 consecutive infants born in the 1967-1979 period suitable for secular trend analysis and case-control study of risk factors; and the B series, including 255,834 consecutive stillborn and liveborn infants of the 1980-1982 period suitable for prevalence rate analysis. Anencephaly was registered in 6.0/10,000 births, A spina bifida aperta in 6.2/10,000 births, and cephalocele in 2.4/10,000 births. A stable secular trend was observed for the frequency of all three neural tube defect types. Spina bifida was more frequent in Chile than in the rest of South America. No differences in prevalence rates were seen between tropical and non tropical areas. Parental consanguinity and environmental prenatal factors including maternal illnesses, drug intake, and radiation exposure were found in association with anencephaly and spina bifida.

Epidemiology of ambiguous genitalia in South America.

We present descriptive epidemiologic data on ambiguous genitalia (AG), obtained in the period 1967-1982 by the Latin American Collaborative Study of Congenital Malformations (ECLAMC) through 70 participating hospitals located in 34 cities of 9 South American countries. The observed prevalence rate for isolated AG was about 1/20,000 births, with at least 1/4 and probably as much as 1/2 due to congenital adrenal hyperplasia. When AG cases associated with other congenital anomalies were also considered, the overall frequency of AG was 1/6,900 total births (1/10,000 for live and 1/350 for stillbirths). The prevalence rates were homogeneously distributed among the different countries, and there was a stable secular trend. AG was more frequent in nontropical than in tropical areas of South America, and there was a significant seasonal variation with its acrophase in August (winter). A case-control analysis of 14 risk factors showed an association of AG with a prenatal history of acute maternal illnesses. These data do not support an etiological relationship between AG and hypospadias.

Effect of paternal age in achondroplasia, thanatophoric dysplasia, and osteogenesis imperfecta.

The paternal ages of nonfamilial cases of achondroplasia (AC) (n = 78), thanatophoric dysplasia (TD) (n = 64), and osteogenesis imperfecta (OI) (n = 106), were compared with those of matched controls, from an Italian Indagine Policentrica Italiana sulle Malformazioni Congenite and a South American Estudio Colaborativo Latinoamericano de Malformaciones Congénitas series. The degree of paternal age effect on the origin of these dominant mutations differed among the three conditions. Mean paternal age was highly elevated in AC, 36.30 +/- 6.74 years in the IPIMC, and 37.19 +/- 10.53 years in the ECLAMC; less consistently elevated in TD, 33.60 +/- 7.08 years in the IPIMC, and 36.41 +/- 9.38 years in the ECLAMC; and only slightly elevated in OI in the ECLAMC, 31.15 +/- 9.25 years, but not in the IPIMC, 32.26 +/- 6.07 years. Increased maternal age or birth order in these conditions disappeared when corrected for paternal age. Approximately 50% of AC and TD cases, and only 30% of OI cases, were born to fathers above age 35 years. For AC and TD, the increase in relative incidence with paternal age fitted an exponential curve. The variability of paternal age effect in these new mutations could be due, among other reasons, to the high proportion of germ-line mosaicism in OI parents, or to the localization of the AC gene, mapped to the 4p16.3 region, in the neighborhood of an unstable DNA area.

Pectoralis major muscle defect and Poland complex.

Pectoralis major muscle defect (PMD) was diagnosed in 27 infants from a series of 599,109 live births in South America (1/22,189). In all 27 cases the PMD was unilateral, mainly affecting the right side (20/27), and there were more male (19/27) than female cases. No familial cases and no parental consanguinity were recorded. A positive correlation was observed between PMD and sex hormone intake and vaginal bleeding in the first trimester of pregnancy. In 12 (1/49,925) of the 27 PMD cases hypoplasia and/or syndactyly of the ipsilateral hand was also diagnosed. The index-middle interdigital space was affected in all 11 cases with symbrachydactyly. Additional congenital anomalies were observed in 4/27 cases, and they were: hemangiomas, hypospadias, and clubfeet. Poland complex (12 cases), isolated PMD (15 cases), and isolated symbrachydactyly (18 cases), showed a similar pattern for symmetry, sidedness, syndactyly type, and sex ratio.

On monitoring the multiply malformed infant. I: Case-finding, case-recording, and data handling in a Latin American program.

The methods used by the Latin American Collaborative Study of Congenital Malformations (ECLAMC) for monitoring the birth prevalence of multiply malformed infants are based on a clinical-epidemiological approach oriented to the early detection of teratogenic agents. They consist of three steps: 1) the analysis of observed vs expected rates of all congenital anomalies (CA), including their isolated and associated forms; 2) the same type of analysis applied to each multiple congenital anomaly (MCA) pattern; and 3) a clinical case presentation reserved only for those considered as true MCA because of presence of three or more independent CA. During the period 1982-1983 299,231 infants were examined. Multiply malformed infants, excluding Down syndrome cases, were born at a rate of 4/10,000, 40% having syndromes (two or more interrelated CA), 30% anomaly pairs, ie, two independent CA, and 30% true MCA cases. In a program with 150,000 births per year, as in ECLAMC, this means about five true MCA cases per week, a number easily handled individually on a clinical basis.

Isolated small intestinal atresias in Latin America and Spain: epidemiological analysis.

Stenosis, atresia, or absence of part of the duodenum, jejunum, or ileum are generally considered small intestinal atresias (SIAs). SIAs occur as isolated defects, in combination with other unrelated congenital anomalies, or as part of syndromes. We performed an epidemiological study of infants with isolated SIAs using data from two large congenital defects registries, one from Latin America (ECLAMC) and the other from Spain (ECEMC). The overall prevalence of SIAs is similar in both programs, being 1.32 per 10,000 livebirths in Spain and 1.29 per 10.000 livebirths in Latin America. Our results suggest that infants with isolated SIAs are characterized epidemiologically on the basis of shorter gestational age and low birthweight, an association with twinning, the parents are more frequently consanguineous, and their pregnancies are more frequently complicated by vaginal bleeding. The results also suggest an association between some maternal infections and ileal atresia. The fact that these characteristics have been observed in children with these types of anomalies occurring in different geographical areas and populations supports the conclusion that these characteristics are causally related to these defects.

Survival of children with Down syndrome in South America. ECLAMC-Downsurv Group. Latin American Collaborative Study of Congenital Malformations.

The first step of all healthcare actions aimed at promoting an appropriate quality of life for infants affected by Down syndrome (DS) is to ensure their survival. This investigation was aimed at estimating the infant mortality rate of infants affected with DS in urban populations of South America. Thirty-three hospitals included in the Latin American Collaborative Study of Congenital Malformations (ECLAMC) distributed in 23 cities of 5 South American countries followed 360 liveborn DS cases born during the 1988-1992 period. Families were recontacted after the infant should have reached the age of one year. The collected data included information about health status; i.e., frequency and dates of diagnosed illnesses and hospital admissions, and, in case of death, information on date, place and cause of death, and illness immediately before death. Information about the interviews included place, date, and name of the interviewer. A closed questionnaire was employed by the interviewers, mostly physicians, nurses, and social workers. Life table analysis up to the age of one year was performed by the actuarial survival method. The overall mean survival at age one year was 0.736 (SE=0.023). Thirty-three (9.2%) of the 360 cases died neonatally, and 62 (17.2%) within the remaining 2-to-12-month interval. The probability of survival at one year of age did not differ between public (209 cases; mean 0.718; SE=0.031) and private (151 cases; mean: 0.762; SE=0.035) (chi2:0.87; df:1; P >0.05) health systems. The 150 DS cases with a congenital heart defect (CHD) had a significantly lower P robability of survival at the age of one year (mean: 0.660; SE: 0.039) than did the 210 cases without CHD (mean: 0.790; SE: 0.028) (chi2:6.67; df:1; P <0.01). The death rate in the first year of life for DS cases without a detected cardiac defect (21%) is significantly higher than that reported in developed countries; namely, 16% from Italy, 11% from Canada, 10% from England, and 7% from Denmark.

Prevalence rates of microtia in South America.

A high frequency of microtia in Quito (Ecuador) was detected by the Latin American Collaborative Study of Congenital Malformations (ECLAMC), and the data available on file were used to define the cluster and to test some potential risk factors. The Quito material consisted of 46 041 livebirths from two hospitals. The rest of the sample consisted of 553 068 livebirths from 58 hospitals located in 24 cities of six other South American countries. The observed prevalence rate for microtia was over five times higher in Quito (17.4/10 000) than in the other cities (3.2/10 000). The microtias from Quito were mostly represented by isolated forms (without other anomalies except for preauricular tags and/or sinuses), and the proportion of severe microtia was higher than in the rest of the sample. Neither seasonal, nor secular variations were observed in Quito or the rest of the sample; data suggesting the geographical cluster did not arise in recent years. Case-control analysis of familial, prenatal and perinatal history data in 184 cases with isolated microtia (68 from Quito and 116 from the rest) and in 184 matched non-malformed control newborn babies, identified prenatal drug exposure, high birth order, and elevated paternal age as risk factors for the microtias born in Quito.

Monthly and seasonal variations in the frequency of congenital anomalies.

Three large and comparable series of births were used to test the working hypothesis that if there is a real seasonal variation in the frequency of a given congenital malformation; it would have to be shown by adequate analysis; to be more overt in non-tropical areas; and to be six months out of phase in northern and southern hemispheres. The data set were hospital births from tropical (287,165 births) and non-tropical (582,585 births) South America, and from Italy (508,536 births). Sixteen well-defined malformation types were tested: anencephaly, spina bifida, cephalocoele, hydrocephaly, microtia, cleft palate, cleft lip, oesophageal atresia, anal atresia, hypospadias, pes equino-varus, pes talovalgus, postaxial polydactylyl, pre-axial polydactylyl, diaphragmatic hernia, and Down's syndrome. No seasonal variation was proven (p less than 0.01) for any malformation type in any of the three series of data by means of Walter and Elwood's test, or Hewitt et al's non-parametric test2 applied to seven instances with sample sizes smaller than 50 cases. Variations of borderline significance (p less than 0.05) included oesophageal atresia in tropical South America, none in non-tropical South America, and anencephaly in Italy. It is concluded that seasonal variation in the occurrence of congenital malformations is a rare phenomenon when tests are strictly used within their recommended limitations.

MSX1 and TGFB3 contribute to clefting in South America.

MSX1 and TGFB3 have been proposed as genes in which mutations may contribute to non-syndromic forms of oral clefts; however, an interaction between these genes has not been described. The present study attempts to detect transmission distortion of MSX1 and TGFB3 in 217 South American children from their respective mothers. With transmission disequilibrium test analysis, cleft lip with/without cleft palate, cleft lip with palate plus cleft palate only, and all datasets combined showed evidence of association with MSX1 (p = 0.004, p = 0.037, and p = 0.001, respectively). With likelihood ratio test analysis, "cleft lip only" showed association with MSX1 (p = 0.04) and "cleft palate only" with TGFB3 (p = 0.02). A joint analysis of MSX1 and TGFB3 suggested that there may be an interaction between these two loci to increase cleft susceptibility. These results suggest that MSX1 and TGFB3 mutations make a contribution to clefts in South American populations.

Pregnancy outcome after exposure to misoprostol in Brazil: a prospective, controlled study.

BACKGROUND: Misoprostol, a synthetic prostaglandin E1 analog is labeled for the treatment of gastric and duodenal ulcers. In Brazil, where abortion is not a legal procedure, there is a widespread popular misuse of this drug in abortion attempts. This misuse and the fact that, in many cases the desired pregnancy termination does not occur, raise concerns about fetal safety. Case reports of congenital anomalies after maternal use of misoprostol have been published. The objective of this work was to compare pregnancy outcome following misoprostol exposure with a matched control group. This is the first prospective controlled study on fetal safety after misoprostol use. METHODS: A prospective, observational cohort study with 86 exposed and 86 pair-matched, non-exposed controls. RESULTS: There was no significant difference in the rates of major or minor birth between exposed compared to non-exposed infants (2/67 vs 2/81, major defects; 7/67 vs. 3/81, minor anomalies) There were significantly more miscarriages in the exposed group (17.1% vs. 5.8%; relative risk, 2.97; 95% confidence interval, 1.12 to 7.88). There was no statistical difference in gestational age at delivery, birth weight, sex ratio, rate of prematurity, low birth weight, or rates of cesarean section between groups. CONCLUSIONS: Our study, despite its limited statistical power, does not suggest a potent teratogenic action of misoprostol exposure during pregnancy.

Association of AXIN2 with non-syndromic oral clefts in multiple populations.

We have previously shown the association of AXIN2 with oral clefts in a US population. Here, we expanded our study to explore the association of 11 AXIN2 markers in 682 cleft families from multiple populations. Alleles for each AXIN2 marker were tested for transmission distortion with clefts by means of the Family-based Association Test. We observed an association with SNP rs7224837 and all clefts in the combined populations (p = 0.001), and with SNP rs3923086 and cleft lip and palate in Asian populations (p = 0.004). We confirmed our association findings in an additional 528 cleft families from the United States (p < 0.009). We tested for gene-gene interaction between AXIN2 and additional cleft susceptibility loci. We assessed and detected Axin2 mRNA and protein expression during murine palatogenesis. In addition, we also observed co-localization of Axin2 with Irf6 proteins, particularly in the epithelium. Our results continue to support a role for AXIN2 in the etiology of human clefting. Additional studies should be performed to improve our understanding of the biological mechanisms linking AXIN2 to oral clefts.

Novel cleft susceptibility genes in chromosome 6q.

Cleft lip/palate is a defect of craniofacial development. In previous reports, chromosome 6q has been suggested as a candidate region for cleft lip/palate. A multipoint posterior probability of linkage analysis of multiplex families from the Philippines attributed an 88% probability of harboring a cleft-susceptibility gene to a narrower region on bands 6q14.2-14.3. We genotyped 2732 individuals from families and unrelated individuals with and without clefts to investigate the existence of possible cleft-susceptibility genes in this region. We found association of PRSS35 and SNAP91 genes with cleft lip/palate in the case-control cohort and in Caucasian families. Haplotype analyses support the individual associations with PRSS35. We found Prss35 expression in the head and palate of mouse embryos at critical stages for palatogenesis, whereas Snap91 was expressed in the adult brain. We provide further evidence of the involvement of chromosome 6q in cleft lip/palate and suggest PRSS35 as a novel candidate gene.