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.

Polymorphisms in the fetal progesterone receptor and a calcium-activated potassium channel isoform are associated with preterm birth in an Argentinian population.

OBJECTIVE: To investigate genetic etiologies of preterm birth (PTB) in Argentina through evaluation of single-nucleotide polymorphisms (SNPs) in candidate genes and population genetic admixture. STUDY DESIGN: Genotyping was performed in 389 families. Maternal, paternal and fetal effects were studied separately. Mitochondrial DNA (mtDNA) was sequenced in 50 males and 50 females. Y-chromosome anthropological markers were evaluated in 50 males. RESULT: Fetal association with PTB was found in the progesterone receptor (PGR, rs1942836; P=0.004). Maternal association with PTB was found in small conductance calcium activated potassium channel isoform 3 (KCNN3, rs883319; P=0.01). Gestational age associated with PTB in PGR rs1942836 at 32-36 weeks (P=0.0004). MtDNA sequencing determined 88 individuals had Amerindian consistent haplogroups. Two individuals had Amerindian Y-chromosome consistent haplotypes. CONCLUSION: This study replicates single locus fetal associations with PTB in PGR, maternal association in KCNN3, and demonstrates possible effects for divergent racial admixture on PTB.

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.

Brazil: public health genomics.

Brazil represents half of South America and one third of Latin America, having more than 186 million inhabitants. After China and India it is the third largest developing country in the world. The wealth is unequally distributed among the states and among the people. Brazil has a large and complex health care system. A Universal Public Health System (SUS: Sistema SPACEnico de Saúde) covers the medical expenses for 80% of the population. The genetic structure of the population is very complex, including a large proportion of tri- hybrid persons, genetic isolates, and a panmictic large majority. Genetic services are offered at 64 genetic centers, half of them public and free. Nationwide networks are operating for inborn errors of metabolism, oncogenetics, and craniofacial anomalies. The Brazilian Society of Medical Genetics (SBGM) has granted 120 board certifications since 1986, and 7 recognized residences in medical genetics are operating in the country. Three main public health actions promoted by the federal government have been undertaken in the last decade, ultimately aimed at the prevention of birth defects. Since 1999, birth defects are reported for all 3 million annual live births, several vaccination strategies aim at the eradication of rubella, and wheat and maize flours are fortified with folic acid. Currently, the government distributes over 2 million US dollars to finance 14 research projects aimed at providing the basis for the adequate prevention and care of genetics disorders through the SUS. Continuity of this proactive attitude of the government in the area of genomics in public health is desired.

Reduced folate carrier 1 (RFC1) is associated with cleft of the lip only.

In this report, we have reanalyzed genotyping data in a collection of families from South America based on maternal origin. Genotyping analysis was performed at the Craniofacial Anomalies Research Center at the University of Iowa. These genotypes were derived from genomic DNA samples obtained from blood spots from children born with isolated orofacial clefts in 45 hospitals located in eight countries (Argentina, Bolivia, Brazil, Chile, Ecuador, Paraguay, Uruguay, and Venezuela) collaborating with ECLAMC (Latin American Collaborative Studies of Congenital Malformations) between January 1998 and December 1999. Dried blood samples were sent by regular mail to the Laboratory of Congenital Malformations, Federal University of Rio de Janeiro. Previous findings suggested that mitochondrial haplotype D is more commonly found among cleft cases born in South America. We hypothesized that association of certain genes may depend upon the ethnic origin, as defined by population-specific markers. Therefore, we tested if markers in MTHFR (5,10-methylenetetrahydrofolate reductase) and RFC1 (reduced folate carrier 1) were associated with oral clefts, depending on the maternal origin defined by the mitochondrial haplotype. Transmission distortion of alleles in MTHFR C677T and RFC1 G80A polymorphic variants was tested in 200 mother/affected child pairs taking into consideration maternal origin. RFC1 variation was over-transmitted to children born with cleft lip only (P = 0.017) carrying mitochondrial DNA haplotypes other than haplotype D. Our results provide a new indication that variation in RFC1 may contribute to cleft lip only. Future studies should investigate the association between oral clefts and RFC1 based on more discrete phenotypes.

Reduced folate carrier 1 (RFC1) is associated with cleft of the lip only

In this report, we have reanalyzed genotyping data in a collection of families from South America based on maternal origin. Genotyping analysis was performed at the Craniofacial Anomalies Research Center at the University of Iowa. These genotypes were derived from genomic DNA samples obtained from blood spots from children born with isolated orofacial clefts in 45 hospitals located in eight countries (Argentina, Bolivia, Brazil, Chile, Ecuador, Paraguay, Uruguay, and Venezuela) collaborating with ECLAMC (Latin American Collaborative Studies of Congenital Malformations) between January 1998 and December 1999. Dried blood samples were sent by regular mail to the Laboratory of Congenital Malformations, Federal University of Rio de Janeiro. Previous findings suggested that mitochondrial haplotype D is more commonly found among cleft cases born in South America. We hypothesized that association of certain genes may depend upon the ethnic origin, as defined by population-specific markers. Therefore, we tested if markers in MTHFR (5,10-methylenetetrahydrofolate reductase) and RFC1 (reduced folate carrier 1) were associated with oral clefts, depending on the maternal origin defined by the mitochondrial haplotype. Transmission distortion of alleles in MTHFR C677T and RFC1 G80A polymorphic variants was tested in 200 mother/affected child pairs taking into consideration maternal origin. RFC1 variation was over-transmitted to children born with cleft lip only (P = 0.017) carrying mitochondrial DNA haplotypes other than haplotype D. Our results provide a new indication that variation in RFC1 may contribute to cleft lip only. Future studies should investigate the association between oral clefts and RFC1 based on more discrete phenotypes.

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.

Contributions of PTCH gene variants to isolated cleft lip and palate.

OBJECTIVE: Mutations in patched (PTCH) cause the nevoid basal cell carcinoma syndrome (NBCCS), or Gorlin syndrome. Nevoid basal cell carcinoma syndrome may present with developmental anomalies, including rib and craniofacial abnormalities, and predisposes to several tumor types, including basal cell carcinoma and medulloblastoma. Cleft palate is found in 4% of individuals with nevoid basal cell carcinoma syndrome. Because there might be specific sequence alterations in PTCH that limit expression to orofacial clefting, a genetic study of PTCH was undertaken in cases with cleft lip and/or palate (CL/P) known not to have nevoid basal cell carcinoma syndrome. RESULTS: Seven new normal variants spread along the entire gene and three missense mutations were found among cases with cleft lip and/or palate. One of these variants (P295S) was not found in any of 1188 control samples. A second variant was found in a case and also in 1 of 1119 controls. The third missense (S827G) was found in 5 of 1369 cases and in 5 of 1104 controls and is likely a rare normal variant. Linkage and linkage desequilibrium also was assessed using normal variants in and adjacent to the PTCH gene in 220 families (1776 individuals), each with two or more individuals with isolated clefting. Although no statistically significant evidence of linkage (multipoint HLOD peak = 2.36) was uncovered, there was borderline evidence of significant transmission distortion for one haplotype of two single nucleotide polymorphisms located within the PTCH gene (p = .08). CONCLUSION: Missense mutations in PTCH may be rare causes of isolated cleft lip and/or palate. An as yet unidentified variant near PTCH may act as a modifier of cleft lip and/or palate.

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.

Inheritance of cleft palate in South America: evidence for a major locus recessive.

OBJECTIVES: Determine the model of inheritance of non-syndromic cleft palate in humans. DESIGN: Complex segregation analysis performed in families of consecutive newborns affected with non-syndromic cleft palate. SETTING AND SAMPLE POPULATION: The Latin American Collaborative Study of Congenital Malformations (ECLAMC). Four hundred and seven consecutive newborns affected with non-syndromic cleft palate registered during the period 1967-97. OUTCOME MEASURE: Likelihood ratio test and Akaike information criterion (AIC) values. RESULTS: The single major locus recessive model provided a significantly better explanation of the data. It was the most parsimonious and had the smallest AIC value of the six models tested with approximately the same likelihood as the general model (chi2 = 2.44, p = 0.5). CONCLUSIONS: To have defined a genetic model for non-syndromic cleft palate and provided evidence for a single major locus inheritance suggests that genetic linkage studies could be implemented.

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.

Genetic origins in a South American clefting population.

It has been proposed that susceptibility to clefting in South America is related to Amerindian ancestry, where clefting is present at a higher frequency than in the other admixed populations (Caucasian and African) that make up the diverse racial mix of current South Americans. To clarify the genetic origins and establish a method for genetic mapping, mitochondrial DNA variation and Y-chromosome markers were studied in a South American population affected with clefting. Two-hundred and seventeen subjects and matched controls were selected through the Latin-American Collaborative Study of Congenital Malformations (ECLAMC). The case group showed a higher frequency of Native American haplogroups and a lower frequency of African haplogroups (p < 0.00001). In addition, the case group showed a much higher frequency of the specific native American haplogroup D than the control group (p < 0.00001). For the Y-chromosome markers, the case group showed a lower frequency of the African-specific marker, YAP (p = 0.002), and a higher frequency of the Native American-specific marker, DYS199 (p < 0.00001). Even though differences were found in the frequencies of the markers studied, the contribution of each founder population was similar for both groups. Results suggest a strong Native American maternal contribution and a strong Caucasian (Spanish and Portuguese) paternal contribution to the population studied. The implications of this finding include the possibility of using admixture mapping approaches to this population.

Epidemiological methods to assess the correlation between industrial contaminants and rates of congenital anomalies.

The present knowledge of epidemiological methods, applied to assess the correlation between industrial contaminants and rates of congenital anomalies is reviewed. The concept of congenital anomalies may be extended to include other adverse reproductive outcomes, such as malformations, infant mortality, stillbirths, spontaneous abortions, intrauterine growth retardation, ectopic pregnancies, multiple births, altered secondary sex ratio, and parental sub-fertility. The review of occupational exposures associated with congenital anomalies indicated: (1) inconsistency of the reported associations; (2) more positive than negative associations; (3) solvents are the best studied, and the most frequently reported teratogenic chemicals; (4) common congenital anomalies are the most frequently studied diagnostic categories, while other defects are grouped into larger categories, with little biological meaning. The review of environmental exposures indicated that: (1) single-site studies outnumber multi-site ones; (2) results are heterogeneous; (3) congenital anomalies are, in general, unspecific, and grouped into large categories, such as those defined by anatomic systems. Recent developments in molecular biology anticipate the possibility to measure exposures directly, instead of by different "proxies", as well as to analyze the genetic predisposition for the teratogenic response to given environmental agents. The strategy of building up large banks of biological materials has already started in several birth defects registries. The following procedural guidelines to assess the teratogenicity of a pollutant are recommended: (1) strength of the association; (2) consistency of findings in different studies; (3) specificity of the association; (4) time-exposure relationship; (5) existence of a dose-response gradient between exposure and disease occurrence; (6) biological plausibility; (7) coherence of the evidence with natural history of the disease; (8) experimental (or quasi-experimental) evidence and (9) reasoning by analogy.

Search for ethnic, geographic, and other factors in the epidemiology of Down syndrome in South America: analysis of data from the ECLAMC project, 1967-1997.

We have analyzed data on 3,157 cases of Down syndrome (DS) from nine South American countries in consecutive series of hospital live births over a 30-year period, with particular emphasis on possible ethnic or geographic variations in maternal age-adjusted incidence. The data constitute the largest series of DS cases assembled to date from an area lacking advanced health care systems. Absolute incidence rates were estimated from total hospital live births; relative rates were estimated from matched case-control data using conditional logistic regression. Maternal age-adjusted rates were closely similar to those reported elsewhere, and showed little or no dependency on other factors investigated, including paternal age, birth order, ancestral origin, country of birth, maternal educational level, maternal ABO and Rhesus blood groups, interval to and outcome of mother's previous pregnancy, and parental consanguinity. The absence of an effect of high birth order was particularly notable because of the relatively large number of grand multipara resulting from high fertility in this population. The study adds to a body of evidence suggesting that maternal age-adjusted DS rates vary little across human populations, and are therefore unlikely to be greatly influenced by genetic or environmental factors that differ between them. An unusual finding was of a markedly lower sex ratio (98 males per 100 females) than has been reported in other DS samples.

Identification of novel mutations in SHH and ZIC2 in a South American (ECLAMC) population with holoprosencephaly.

Holoprosencephaly (HPE) is genetically heterogeneous with four genes, SIX3, SHH, TGIF, and ZIC2 that have been identified to date and that are altered in 12% of patients. To analyze this prevalence in a South American population-based sample (57 HPE cases in 244,511 live and still births or 1 in 4300), we performed a mutational study of these genes in 30 unrelated children (26 newborns and 4 non-newborns) with HPE being ascertained by ECLAMC (Latin American Collaborative Study of Congenital Malformations). We identified three novel mutations: two were missense mutations of the SHH gene (Cys183-->Phe; His140-->Pro); the third mutation was a 2-bp deletion in the zinc-finger region of the ZIC2 gene. These molecular results explained 8% (2/26 newborn samples) of the HPE cases in this South American population-based sample, a proportion similar to our previously published data from a collection of cases.

Parental consanguinity in specific types of congenital anomalies.

Parental consanguinity, as a recognized risk factor for congenital anomalies, has mainly been studied with a focus on the types of parental relationships and their effects on genetic syndromes or birth defects in general. The present work analyzed the association between parental consanguinity and congenital anomalies, split, when possible, into clinical subtypes, in an attempt to obtain some insight into their recognized etiological heterogeneity. The material consisted of 34,102 newborn infants, affected by one of 47 selected congenital anomaly types, ascertained by the Latin-American Collaborative Study of Congenital Malformations (ECLAMC) during the period from 1967 to 1997. The consanguinity rate for each congenital anomaly type was compared with that of the population under study (0.96%), and the potentially confounding effect of six selected variables was controlled through a conditional logistic regression analysis for those congenital anomalies significantly associated with consanguinity. Pre-occurrence rates for the same congenital anomaly in sibships of consanguineous and non-consanguineous cases were compared. A significant association with parental consanguinity was observed for three congenital anomaly types: hydrocephalus, postaxial hand polydactyly, and bilateral cleft lip +/- cleft palate, while three additional anomalies, namely, cephalocele, microcephaly, and hand + foot postaxial polydactyly, showed a positive association, but statistical significance disappeared after adjustment for confounders, probably owing to sample size reduction. The association between consanguinity and Down syndrome was mainly due to the confounding effect of maternal age, while for hydrops fetalis and 2-3 toe syndactyly, the observed positive association could not be tested for confounders due to sample size reduction.

[ECLAMC: 30-year study of epidemiological surveillance of neural tube defects in Chile and Latin America]. / ECLAMC: estudio de 30 años de vigilancia epidemiológica de defectos de tubo neural en Chile y en Latinoamérica.

BACKGROUND: There is an urgent need to assess the impact, on the incidence of neural tube defects, of the recently implanted flour fortification with folic acid in Chile. The Collaborative Study of Congenital Malformations in Latin America (ECLAMC) has carried out an uninterrupted register of congenital malformations in the last thirty years. AIM: To assess the epidemiology of neural tube defects in Chile and determine the most adequate base period to evaluate the effects of Folic acid fortification. MATERIAL AND METHODS: Analysis of ECLAMC database that has registered all births over 500 g from 1967. RESULTS: From 1967 to 1999, there were 434.624 births in 18 hospitals in Chile and 3.586.569 births in 155 hospitals in the rest of Latin America. The rate of neural tube defects in Chile was 17.03 per 10.000, significantly higher than the rest of ECLAMC (14.88 per 10.000). The prevalence of neural tube defects has a statistically significant secular tendency to increase in the study period and is higher among stillbirths, newborns with a birth weight of less than 1500 g, women and offspring of mothers aged less than 19 years old. CONCLUSIONS: The period 1982-1999 is considered the best period for reference comparisons of the effects folic acid supplementation.