Mothers of children with Down syndrome: a clinical and epidemiological study.

Down syndrome is the main genetic cause of intellectual disability. Many studies describe the clinical characteristics of DS patients; however, few have investigated the clinical profile of mothers who have children with DS. Advanced maternal age (≥ 35 years old) is a risk factor for DS. Although there is an overall increase in pregnancies among women with advanced maternal age, there is still a lack of awareness of the increased risk of aneuploidy. Here, we reported the clinical and epidemiological profile of DS children and their mothers in a public reference hospital in the State of Rio de Janeiro, Brazil. For data collection, we performed a face-to-face interview guided by a structured questionnaire with closed-ended questions. A total of 344 individuals, 172 mothers and their DS children, were included in this study. Our results show that 56% of the mothers sampled were ≥ 35 years of age at childbirth. Although 98% of them received prenatal care, only 4% obtained a prenatal diagnosis of DS. Most mothers reported not drinking alcohol or smoking cigarettes during pregnancy. Furthermore, 91% of women took prenatal vitamins and supplements; however, 47% were not aware of their benefits for a healthy pregnancy. Given the strict correlation between advanced maternal age and DS, prenatal care should include genetic counseling for women over 35 years of age. This study highlights the importance of prenatal care and the urgent need for better DS screening allowing for immediate postnatal care, positively impacting the life expectancy of these patients.

PVR/CD155 Ala67Thr Mutation and Cleft Lip/Palate.

The 19q13 locus has been linked to cleft lip and palate by our group and independently by others. Here we fine mapped the region in an attempt to identify an etiological variant that can explain cleft lip and palate occurrence. A total of 2739 individuals born with cleft lip and palate, related to individuals born with cleft lip and palate, and unrelated were studied. We used linkage and association approaches to fine map the interval between D19S714 and D19S433 and genotypes were defined by the use of TaqMan chemistry. We confirmed our previous findings that markers in PVR/CD155 are associated with cleft lip and palate. We studied the mutation Ala67Thr further and calculated its penetrance. We also attempted to detect PVR/CD155 expression in human whole saliva. Our results showed that markers in PVR/CD155 are associated with cleft lip and palate and the penetrance of the Ala67Thr is very low (between 1% and 5%). We could not detect PVR/CD155 expression in adult human whole saliva and PVR/CD155 possibly interacts with maternal infection to predispose children to cleft lip only.

Genome-wide meta-analyses of nonsyndromic orofacial clefts identify novel associations between FOXE1 and all orofacial clefts, and TP63 and cleft lip with or without cleft palate.

Nonsyndromic orofacial clefts (OFCs) are a heterogeneous group of common craniofacial birth defects with complex etiologies that include genetic and environmental risk factors. OFCs are commonly categorized as cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP), which have historically been analyzed as distinct entities. Genes for both CL/P and CP have been identified via multiple genome-wide linkage and association studies (GWAS); however, altogether, known variants account for a minority of the estimated heritability in risk to these craniofacial birth defects. We performed genome-wide meta-analyses of CL/P, CP, and all OFCs across two large, multiethnic studies. We then performed population-specific meta-analyses in sub-samples of Asian and European ancestry. In addition to observing associations with known variants, we identified a novel genome-wide significant association between SNPs located in an intronic TP63 enhancer and CL/P (p = 1.16 × 10-8). Several novel loci with compelling candidate genes approached genome-wide significance on 4q21.1 (SHROOM3), 12q13.13 (KRT18), and 8p21 (NRG1). In the analysis of all OFCs combined, SNPs near FOXE1 reached genome-wide significance (p = 1.33 × 10-9). Our results support the highly heterogeneous nature of OFCs and illustrate the utility of meta-analysis for discovering new genetic risk factors.

A multi-ethnic genome-wide association study identifies novel loci for non-syndromic cleft lip with or without cleft palate on 2p24.2, 17q23 and 19q13.

Orofacial clefts (OFCs), which include non-syndromic cleft lip with or without cleft palate (CL/P), are among the most common birth defects in humans, affecting approximately 1 in 700 newborns. CL/P is phenotypically heterogeneous and has a complex etiology caused by genetic and environmental factors. Previous genome-wide association studies (GWASs) have identified at least 15 risk loci for CL/P. As these loci do not account for all of the genetic variance of CL/P, we hypothesized the existence of additional risk loci. We conducted a multiethnic GWAS in 6480 participants (823 unrelated cases, 1700 unrelated controls and 1319 case-parent trios) with European, Asian, African and Central and South American ancestry. Our GWAS revealed novel associations on 2p24 near FAM49A, a gene of unknown function (P = 4.22 × 10-8), and 19q13 near RHPN2, a gene involved in organizing the actin cytoskeleton (P = 4.17 × 10-8). Other regions reaching genome-wide significance were 1p36 (PAX7), 1p22 (ARHGAP29), 1q32 (IRF6), 8q24 and 17p13 (NTN1), all reported in previous GWASs. Stratification by ancestry group revealed a novel association with a region on 17q23 (P = 2.92 × 10-8) among individuals with European ancestry. This region included several promising candidates including TANC2, an oncogene required for development, and DCAF7, a scaffolding protein required for craniofacial development. In the Central and South American ancestry group, significant associations with loci previously identified in Asian or European ancestry groups reflected their admixed ancestry. In summary, we have identified novel CL/P risk loci and suggest new genes involved in craniofacial development, confirming the highly heterogeneous etiology of OFCs.

Aquaporin 5 Interacts with Fluoride and Possibly Protects against Caries.

Aquaporins (AQP) are water channel proteins and the genes coding for AQP2, AQP5, and AQP6 are clustered in 12q13. Since AQP5 is expressed in serous acinar cells of salivary glands, we investigated its involvement in caries. DNA samples from 1,383 individuals from six groups were studied. Genotypes of eight single nucleotide polymorphisms covering the aquaporin locus were tested for association with caries experience. Interaction with genes involved in enamel formation was tested. The association between enamel microhardness at baseline, after creation of artificial caries lesion, and after exposure to fluoride and the genetic markers in AQP5 was tested. Finally, AQP5 expression in human whole saliva, after exposure to fluoride in a mammary gland cell line, which is known to express AQP5, and in Wistar rats was also verified. Nominal associations were found between caries experience and markers in the AQP5 locus. Since these associations suggested that AQP5 may be inhibited by levels of fluoride in the drinking water that cause fluorosis, we showed that fluoride levels above optimal levels change AQP5 expression in humans, cell lines, and rats. We have shown that AQP5 is involved in the pathogenesis of caries and likely interacts with fluoride.

Role of estrogen related receptor beta (ESRRB) in DFN35B hearing impairment and dental decay.

BACKGROUND: Congenital forms of hearing impairment can be caused by mutations in the estrogen related receptor beta (ESRRB) gene. Our initial linkage studies suggested the ESRRB locus is linked to high caries experience in humans. METHODS: We tested for association between the ESRRB locus and dental caries in 1,731 subjects, if ESRRB was expressed in whole saliva, if ESRRB was associated with the microhardness of the dental enamel, and if ESRRB was expressed during enamel development of mice. RESULTS: Two families with recessive ESRRB mutations and DFNB35 hearing impairment showed more extensive dental destruction by caries. Expression levels of ESRRB in whole saliva samples showed differences depending on sex and dental caries experience. CONCLUSIONS: The common etiology of dental caries and hearing impairment provides a venue to assist in the identification of individuals at risk to either condition and provides options for the development of new caries prevention strategies, if the associated ESRRB genetic variants are correlated with efficacy.

Clinical epidemiology of skeletal dysplasias in South America.

Currently accepted birth prevalence for osteochondrodysplasias (OCD) of about 2/10,000 is based on few studies from small series of cases. We conducted a study based on more than 1.5 million births. OCD cases were detected from 1,544,496 births occurring and examined in 132 hospitals of ECLAMC (Latin American Collaborative Study of Congenital Malformations) between 2000 and 2007. Cases were detected and registered according to a pre-established protocol, and then ranked in four diagnostic evidence levels (DEL), according to available documentation. For the analysis of risk factors, a healthy control sample born in the same period was used. OCD was diagnosed in 492 newborns, resulting in a prevalence per 10,000 of 3.2 (95% CI: 2.9-3.5). Perinatal lethality (stillbirths plus early neonatal deaths) occurred in 50% of cases. Prenatal ultrasound diagnosis was made in 73% of cases (n = 359). Among 211 cases from the best documented group (DEL-1) and according to international classification, 33% of cases fit into the G-25 (osteogenesis imperfecta), 29% in Group-1 (FGFR3), and 8% in Group-18 (Bent bones). The prevalence of the main OCD types were: OI-0.74 (0.61-0.89); thanatophoric dysplasia-0.47 (0.36-0.59); and achondroplasia-0.44 (0.33-0.55). Paternal age (31.2 ± 8.5), parity (2.6), and parental consanguinity rate (5.4%) were higher in cases than in controls (P < 0.001). In conclusion, the OCD overall prevalence of 3.2 per 10,000 found seems to be more realistic than previous estimates. This study also confirmed the high perinatal mortality, and the association with high paternal age, parity, and parental consanguinity rate.

CRISPLD2 variants including a C471T silent mutation may contribute to nonsyndromic cleft lip with or without cleft palate.

OBJECTIVE: To assess the association between nonsyndromic (NS) cleft lip with or without cleft palate (CL(P)) and single-nucleotide polymorphisms (SNPs) within the CRISPLD2 gene (cysteine-rich secretory protein LCCL domain containing 2). DESIGN: Four SNPs within the CRISPLD2 gene domain (rs1546124, rs8061351, rs2326398, rs4783099) were genotyped to test for association via family-based association methods. PARTICIPANTS: A total of 5826 individuals from 1331 families in which one or more family member is affected with CL(P). RESULTS: Evidence of association was seen for SNP rs1546124 in U.S. (p  =  .02) and Brazilian (p  =  .04) Caucasian cohorts. We also found association of SNP rs1546124 with cleft palate alone (CP) in South Americans (Guatemala and ECLAMC) and combined Hispanics (Guatemala, ECLAMC, and Texas Hispanics; p  =  .03 for both comparisons) and with both cleft lip with cleft palate (CLP; p  =  .04) and CL(P) (p  =  .02) in North Americans. Strong evidence of association was found for SNP rs2326398 with CP in Asian populations (p  =  .003) and with CL(P) in Hispanics (p  =  .03) and also with bilateral CL(P) in Brazilians (p  =  .004). In Brazilians, SNP rs8061351 showed association with cleft subgroups incomplete CL(P) (p  =  .004) and unilateral incomplete CL(P) (p  =  .003). Prediction of SNP functionality revealed that the C allele in the C471T silent mutation (overrepresented in cases with CL(P) presents two putative exonic splicing enhancer motifs and creates a binding site AP-2 alpha, a transcription factor involved in craniofacial development. CONCLUSIONS: Our results support the hypothesis that variants in the CRISPLD2 gene may be involved in the etiology of NS CL(P).

Folic acid flour fortification: impact on the frequencies of 52 congenital anomaly types in three South American countries.

The aim of the present investigation was to search for a reduction in birth prevalence estimates of 52 selected types of congenital anomalies, associated with folic acid fortification programs in Chile, Argentina, and Brazil. The material included 3,347,559 total births in 77 hospitals of the three countries during the 1982-2007 period: 596,704 births (17 hospitals) in Chile, 1,643,341 (41 hospitals) in Argentina, and 1,107,514 (19 hospitals) in Brazil. We compared pre- and post-fortification rates within each hospital and the resulting Prevalence Rate Ratios (PRRs) were pooled by country. Statistically significant reductions in birth prevalence estimates after fortification were observed for neural tube defects (NTDs), septal heart defects, transverse limb deficiencies, and subluxation of the hip. However, only the reduction of NTDs appeared to be associated with folic acid fortification and not due to other factors, because of its consistency among the three countries, as well as with previously published reports, and its strong statistical significance. Among the NTDs, the maximum prevalence reduction was observed for isolated cephalic (cervical-thoracic) spina bifida, followed by caudal (lumbo-sacral) spina bifida, anencephaly, and cephalocele. This observation suggests etiologic and pathogenetic heterogeneity among different levels of spina bifida, as well as among different NTD subtypes. We concluded that food fortification with folic acid prevents NTDs but not other types of congenital anomalies.

MTHFR 677C-->T and 1298A-->C polymorphisms in children with Down syndrome and acute myeloid leukemia in Brazil.

Down syndrome (DS) is an important risk factor associated with acute leukemia (AL). The presence of polymorphisms that reduce 5,10-methylenetetrahydrofolate reductase (MTHFR) activity has been linked to the multifactorial leukemogenic process. The authors have conducted a study to test whether 677C-->T and/or 1298A-->C polymorphisms of MTHFR would play an additional role in susceptibility of acute myeloid leukemia (AML) in DS children. They also verified whether any polymorphism in the MTHFR gene was associated with the risk of DS. Genetic polymorphisms determination was carried out in 248 samples from healthy individuals as controls and a total of 115 DS children (65 without leukemia and 50 with AML). The present study failed to reveal any association between these polymorphisms and risk of AML in DS children. The data also indicate that MTHFR polymorphisms are not associated with risk of being a DS child.

Second case of Beare-Stevenson syndrome with an FGFR2 Ser372Cys mutation.

Beare-Stevenson syndrome is characterized by cutis gyrata, acanthosis nigricans, skin furrows, skin tags, craniosynostosis, Crouzonoid-like features in some cases and cloverleaf skull in others, anogenital anomalies, and prominent umbilical stump. Reported causes are an FGFR2 Tyr375Cys mutation in nine cases and an FGFR2 Ser372Cys mutation in one case. Here, we report on a second patient with the FGFR2 Ser372Cys mutation.

Non-Latin European descent could be a requirement for association of NTDs and MTHFR variant 677C > T: a meta-analysis.

There are several studies that have found a positive association between neural tube defects (NTDs) and the common mutation 677C > T of 5,10-methylenetetrahydrofolate reductase (MTHFR), and others that have not found such an association. We updated the meta-analyses of the published data about NTDs and MTHFR 677C > T variant from January 1994 to October 2005 identifying 170 potentially relevant studies. After applying pertinent exclusion criteria, 37 different populations from 32 studies were included in the meta-analysis, with a total of 3,530 cases and 6,296 controls. Further we stratified the data according to geographical region and ethnicity, and produced two separated meta-analyses for non-Latin European and Latin European descent populations. The general (odds ratio 1.41; 95% confidence interval 1.24-1.59), and the non-Latin European meta-analyses (1.62; 1.38-1.90) indicate an association of TT genotype and NTDs; no association was demonstrated for Latin European populations (1.16; 0.95-1.43). The examination of non-Latin European studies revealed that the association of TT genotype with NTD has only been proven for Irish populations, both by case-control studies, and by family-based tests, such as the allele transmission disequilibrium test (TDT).

Functional analysis of mutations in TGIF associated with holoprosencephaly.

Holoprosencephaly (HPE) is the most common structural malformation of the forebrain and face in humans. Our current understanding of the pathogenesis of HPE attempts to integrate genetic susceptibility, evidenced by mutations in the known HPE genes, with the epigenetic influence of environmental factors. Mutations or deletions of the human TGIF gene have been associated with HPE in multiple population cohorts. Here we examine the functional effects of all previously reported mutations, and describe four additional variants. Of the eleven sequence variations in TGIF, all but four can be demonstrated to be functionally abnormal. In contrast, no potentially pathogenic sequence alterations were detected in the related gene TGIF2. These results provide further evidence of a role for TGIF in HPE and demonstrate the importance of functional analysis of putative disease-associated alleles.

PVRL1 variants contribute to non-syndromic cleft lip and palate in multiple populations.

Poliovirus Receptor Like-1 (PVRL1) is a member of the immunoglobulin super family that acts in the initiation and maintenance of epithelial adherens junctions and is mutated in the cleft lip and palate/ectodermal dysplasia 1 syndrome (CLPED1, OMIM #225000). In addition, a common non-sense mutation in PVRL1 was discovered more often among non-syndromic sporadic clefting cases in Northern Venezuela in a previous case-control study. The present work sought to ascertain the role of PVRL1 in the sporadic forms of orofacial clefting in multiple populations. Multiple rare and common variants from all three splice isoforms were initially ascertained by sequencing 92 Iowan and 86 Filipino cases and CEPH controls. Using a family-based analysis to examine these variants, the common glycine allele of the G361V coding variant was significantly overtransmitted among all orofacial clefting phenotypes (P = 0.005). This represented G361V genotyping from over 800 Iowan, Danish, and Filipino families. Among four rare amino acid changes found within the V1 and C1 domains, S112T and T131A were found adjacent to critical amino acid positions within the V1 variable domain, regions previously shown to mediate cell-to-cell and cell-to-virus adhesion. The T131A variant was not found in over 1,300 non-affected control samples although the alanine is found in other species. The serine of the S112T variant position is conserved across all known PVRL1 sequences. Together these data suggest that both rare and common mutations within PVRL1 make a minor contribution to disrupting the initiation and regulation of cell-to-cell adhesion and downstream morphogenesis of the embryonic face.

Reduction of birth prevalence rates of neural tube defects after folic acid fortification in Chile.

To verify whether the decreasing neural tube defects birth prevalence rates in Chile are due to folic acid fortification or to pre-existing decreasing trends, we performed a population survey using a network of Estudio Colaborativo Latino Americano de Malformaciones Congenitas (ECLAMC, Latin American Collaborative Study of Congenital Malformations) maternity hospitals in Chile, between the years 1982 and 2002. Within each maternity hospital, birth prevalence rates of spina bifida and anencephaly were calculated from two pre-fortification periods (1982-1989 and 1990-2000), and from one fortified period (2001-2002). There was no historical trend for spina bifida birth prevalence rates before folic acid fortification, and there was a 51% (minimum 27%, maximum 66%) decrease in the birth prevalence rates of this anomaly in the fortified period. The relative risks of spina bifida were homogeneous among hospitals in the two period comparisons. There was no historical trend for the birth prevalence of anencephaly comparing the two pre-fortified periods, but the relative risks were heterogeneous among hospitals in this comparison. There was a 42% (minimum 10%, maximum 63%) decrease in the birth prevalence rate of anencephaly in the fortified period as compared with the immediately pre-fortified period, with homogeneous relative risks among hospitals. Within the methodological constraints of this study we conclude that the birth prevalence rates for both spina bifida and anencephaly decreased as a result of folic acid fortification, without interference of decreasing secular trends.

Is there a familial link between Down's syndrome and neural tube defects? Population and familial survey.

OBJECTIVE: To verify whether Down's syndrome and neural tube defects arise more often in the same family than expected by chance. DESIGN: Population and familial survey. SETTING: Network of maternity hospitals in the Latin American collaborative study of congenital malformations (ECLAMC) in Argentina, Bolivia, Brazil, Chile, Colombia, Ecuador, Paraguay, Peru, Uruguay, and Venezuela between 1982 and 2000. PROBANDS: 2421 cases of neural tube defects, 952 of hydrocephalus, and 3095 of Down's syndrome registered from a total of 1 583 838 live births and stillbirths. MAIN OUTCOME MEASURES: Observed number of cases of Down's syndrome among siblings of probands with a neural tube defect or hydrocephalus and number expected on the basis of maternal age; observed number of cases of neural tube defects or hydrocephalus among siblings of probands with Down's syndrome and number expected according to the prevalence in the same population. RESULTS: Five cases of Down's syndrome occurred among 5404 pregnancies previous to a case of neural tube defect or hydrocephalus, compared with 5.13 expected after adjustment by maternal age. Twelve cases of neural tube defect or hydrocephalus occurred among 8066 pregnancies previous to a case of Down's syndrome, compared with 17.18 expected on the basis of the birth prevalence for neural tube defects plus hydrocephalus in the same population. CONCLUSION: No association occurred between families at risk of neural tube defects and those at risk of Down's syndrome.

Preliminary data on changes in neural tube defect prevalence rates after folic acid fortification in South America.

Several South American countries are fortifying wheat flour with folic acid. However, only Chile started in 2000 to add 2.2 mg/kg, providing 360 microg daily per capita, an acceptable dosage for preventing the occurrence of some neural tube defect (NTD) cases. ECLAMC (Spanish acronym for the Latin American Collaborative Study of Congenital Malformations) routinely monitoring birth defects in South America since 1976, surveyed the impact of this fortification. Data from 361,374 births occurred in 43 South American hospitals, distributed in five different countries, active throughout the 1999-2001 triennium, were selected from the ECLAMC network. Birth prevalence rates for three different congenital anomalies with similar expected prevalence rates, were surveyed by the Cumulative Sum Method (CUSUM) method. They were NTD, oral clefts (OC), and Down syndrome (DS). Expected values were derived from observations made in 1999, and CUSUM was applied to the consecutive series of 24 months covering years 2000 and 2001. Only one of three congenital anomaly types, NTDs, in only one of five sampled out countries, Chile, showed a significant decrease, of 31%, during the 2000-2001 biennium, corresponding to the birth of the periconceptionally fortified infants. The level of significance (P < 0.001) was reached in the 20th month after fortification started, corresponding to August 2001. This is the first observation of a significant decrease in the occurrence of NTD after folic acid food fortification in a population little influenced by confounders common in the developed world as pre-existing secular decreasing trends, and partially unregistered induced abortions.

Prevención primaria de los defectos congénitos / Primary prevention in the congenital defects

Expone las propuestas del ECLAMC para realizar una eficiente prevención de los defectos congénitos, es decir de los nacimientos defectuosos, en forma detallada y documentada, resumiendo una propuesta de prevención en su último capítulo. Es una obra dirigida a todos los interesados en el tema, pertenezcan o no al sector de salud, condensada en un decálogo para evitar las deficiencias físicas y mentales.