Comprehensive germline mutation analysis and clinical profile in a large cohort of Brazilian xeroderma pigmentosum patients.

BACKGROUND: Xeroderma pigmentosum (XP) patients present a high risk of developing skin cancer and other complications at an early age. This disease is characterized by mutations in the genes related to the DNA repair system. OBJECTIVES: To describe the clinical and molecular findings in a cohort of 32 Brazilian individuals who received a clinical diagnosis of XP. METHODS: Twenty-seven families were screened for germline variants in eight XP-related genes. RESULTS: All patients (N = 32) were diagnosed with bi-allelic germline pathogenic or potentially pathogenic variants, including nine variants previously undescribed. The c.2251-1G>C XPC pathogenic variant, reported as the founder mutation in Comorian and Pakistani patients, was observed in 15 cases in homozygous or compound heterozygous. Seven homozygous patients for POLH/XPV variants developed their symptoms by an average age of 7.7 years. ERCC2/XPD, DDB2/XPE and ERCC5/XPG variants were found in a few patients. Aside from melanoma and non-melanoma skin tumours, a set of patients developed skin sebaceous carcinoma, leiomyosarcoma, angiosarcoma, mucoepidermoid carcinoma, gastric adenocarcinoma and serous ovarian carcinoma. CONCLUSIONS: We reported a high frequency of XPC variants in 32 XP Brazilian patients. Nine new variants in XP-related genes, unexpected non-skin cancer lesions and an anticipation of the clinical manifestation in POLH/XPV cases were also described.

p53 signaling pathway polymorphisms associated to recurrent pregnancy loss.

The p53 protein is known for performing essential functions in the maintenance of genomic stability in somatic cells and prevention of tumor formation. Studies of the p53 signaling pathway have suggested associations between some polymorphisms and infertility, post-in vitro fertilization implantation failure and recurrent abortions. The TP53 Pro72Arg polymorphism has been implicated as a risk factor for recurrent pregnancy loss (RPL); however, the association is controversial. In this study, our objective was to evaluate selected polymorphisms in genes of the p53 signalling pathway [TP53 c.215G>C (Pro72Arg), MDM2 c.14+309T>G (SNP309) and LIF c.1414T>G in the region 3' UTR] and determine their effect as risk factors for RPL. In a case-control study, we investigated 120 women with two or more pregnancy losses and 143 fertile control women reporting at least two live births and no history of pregnancy loss. When analyzed separately, the allele and genotype distributions of the polymorphisms in the two groups were not different. However, in a multivariate analysis adjusted for alcohol consumption, smoking, ethnicity, and number of pregnancies, the interaction between the genotypes TP53 Arg/Arg (rs1042522) and MDM2 TT (rs2279744) showed to be associated to RPL, increasing the risk for this condition (OR = 2.58, 95% CI: 1.31-5.07, p = 0.006). In conclusion, our study indicates that the combination of TP53 Arg/Arg (rs1042522) and MDM2 TT (rs2279744) genotypes may be a risk factor for RPL.

Prevalence of ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms in Brazilian breast cancer-unaffected women

Polymorphisms of hormone receptor genes have been linked to modifications in reproductive factors and to an increased risk of breast cancer (BC). In the present study, we have determined the allelic and genotypic frequencies of the ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms and investigated their relationship with mammographic density, body mass index (BMI) and other risk factors for BC. A consecutive and unselected sample of 750 Brazilian BC-unaffected women enrolled in a mammography screening program was recruited. The distribution of PGR PROGINS genotypic frequencies was 72.5, 25.5 and 2.0% for A1A1, A1A2 and A2A2, respectively, which was equivalent to that encountered in other studies with healthy women. The distribution of ERα genotypes was: ERα-397 PvuII C/T: 32.3% TT, 47.5% TC, and 20.2% CC; ERα-351 XbaI A/G: 46.3% AA, 41.7% AG and 12.0% GG. ERα haplotypes were 53.5% PX, 14.3% Px, 0.3% pX, and 32.0% px. These were significantly different from most previously published reports worldwide (P < 0.05). Overall, the PGR PROGINS genotypes A2A2 and A1A2 were associated with fatty and moderately fatty breast tissue. The same genotypes were also associated with a high BMI in postmenopausal women. In addition, the ERα-351 XbaI GG genotype was associated with menarche ≥12 years (P = 0.02). ERα and PGR polymorphisms have a phenotypic effect and may play an important role in BC risk determination. Finally, if confirmed in BC patients, these associations could have important implications for mammographic screening and strategies and may be helpful to identify women at higher risk for the disease.

TP53 PIN3 and PEX4 polymorphisms and infertility associated with endometriosis or with post-in vitro fertilization implantation failure.

p53 has a crucial role in human fertility by regulating the expression of leukemia inhibitory factor (LIF), a secreted cytokine critical for blastocyst implantation. To examine whether TP53 polymorphisms may be involved with in vitro fertilization (IVF) failure and endometriosis (END), we have assessed the associations between TP53 polymorphism in intron 2 (PIN2; G/C, intron 2), PIN3 (one (N, non-duplicated) or two (D, duplicated) repeats of a 16-bp motif, intron 3) and polymorphism in exon 4 (PEX4; C/G, p.P72R, exon 4) in 98 women with END and 115 women with post-IVF failure. In addition, 134 fertile women and 300 women unselected with respect to fertility-related features were assessed. TP53 polymorphisms and haplotypes were identified by amplification refractory mutation system polymerase chain reaction. TP53 PIN3 and PEX4 were associated with both END (P=0.042 and P=0.007, respectively) and IVF (P=0.004 and P=0.009, respectively) when compared with women both selected and unselected for fertility-related features. Haplotypes D-C and N-C were related to higher risk for END (P=0.002, P=0.001, respectively) and failure of IVF (P=0.018 and P=0.002, respectively) when compared with the Fertile group. These results support that specific TP53 haplotypes are associated with an increased risk of END-associated infertility and with post-IVF failure.

High twinning rate in Cândido Godói: a new role for p53 in human fertility.

BACKGROUND: Cândido Godói (CG) is a small town in South Brazil, which has the highest prevalence of twin births in Brazil. Recently, a number of studies have shown that p53 plays an important role in reproduction through blastocyst implantation and intra utero embryo survival. Thus, gene polymorphisms in the p53 pathway were investigated in this population. METHODS: Single nucleotide polymorphisms from five genes in the p53 pathway were investigated, as well as background characteristics of 42 mothers of twins (cases) and 101 mothers of singletons (controls), all residents from CG. RESULTS: Mothers of twins have higher number of pregnancies and higher frequencies of P72 allele at TP53 and T allele at MDM4 genes compared with controls. Logistic regression shows that both TP53 and number of pregnancies maintained their association with twinning (P =0.004 and P =0.002, respectively), with TP53 having a higher odds ratio than number of pregnancies (2.73 versus 1.70, respectively). No interactive effect between TP53 and MDM4 (P =0.966) is observed. As expected, mothers of twins have three times more cases of cancer in their first-degree relatives than control mothers (P =0.011). CONCLUSIONS: Our results suggest that the P72 allele of TP53 is a strong risk factor for twinning in CG, while the number of pregnancies and the T allele at MDM4 may represent weaker risk factors. These two alleles are associated with infertility, but the anti-apoptotic effect of low levels of p53 in general, and of the P72 allele in particular, may play a role after implantation, enhancing the chance for a double pregnancy to succeed to term.

Prevalence of ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms in Brazilian breast cancer-unaffected women.

Polymorphisms of hormone receptor genes have been linked to modifications in reproductive factors and to an increased risk of breast cancer (BC). In the present study, we have determined the allelic and genotypic frequencies of the ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms and investigated their relationship with mammographic density, body mass index (BMI) and other risk factors for BC. A consecutive and unselected sample of 750 Brazilian BC-unaffected women enrolled in a mammography screening program was recruited. The distribution of PGR PROGINS genotypic frequencies was 72.5, 25.5 and 2.0% for A1A1, A1A2 and A2A2, respectively, which was equivalent to that encountered in other studies with healthy women. The distribution of ERα genotypes was: ERα-397 PvuII C/T: 32.3% TT, 47.5% TC, and 20.2% CC; ERα-351 XbaI A/G: 46.3% AA, 41.7% AG and 12.0% GG. ERα haplotypes were 53.5% PX, 14.3% Px, 0.3% pX, and 32.0% px. These were significantly different from most previously published reports worldwide (P < 0.05). Overall, the PGR PROGINS genotypes A2A2 and A1A2 were associated with fatty and moderately fatty breast tissue. The same genotypes were also associated with a high BMI in postmenopausal women. In addition, the ERα-351 XbaI GG genotype was associated with menarche ≥ 12 years (P = 0.02). ERα and PGR polymorphisms have a phenotypic effect and may play an important role in BC risk determination. Finally, if confirmed in BC patients, these associations could have important implications for mammographic screening and strategies and may be helpful to identify women at higher risk for the disease.

Prevalence of the STK15 F31I polymorphism and its relationship with mammographic density

Several studies have identified the single nucleotide polymorphism STK15 F31I as a low-penetrance risk allele for breast cancer, but its prevalence and risk association in the Brazilian population have not been determined. The goal of this study was to identify the frequency of this polymorphism in the Brazilian setting. Considering the high degree of admixture of our population, it is of fundamental importance to validate the results already reported in the literature and also to verify the relationship between this variant and breast cancer risk. A total of 750 women without breast cancer were genotyped using the TaqMan PCR assay for STK15 F31I polymorphism. Clinical information was obtained from review of the medical records and mammographic density from the images obtained using the BI-RADS System. The estimated risk of developing cancer was calculated according to the Gail model. The genotypic frequencies observed in this study were 4.5, 38.7, and 56.6 percent, respectively, for the STK15 F31I AA, AT and TT genotypes. The AT and AA genotypes were encountered significantly more often in premenopausal women with moderately dense, dense and heterogeneously dense breast tissue (P = 0.023). In addition, the presence of the TT genotype was significantly associated with age at menarche ≥12 years (P = 0.023). High mammographic density, associated with increased breast cancer risk, was encountered more frequently in premenopausal women with the risk genotypes STK15 F31I AA and AT. The genotypic frequencies observed in our Brazilian sample were similar to those described in other predominantly European populations.

Prevalence of the STK15 F31I polymorphism and its relationship with mammographic density.

Several studies have identified the single nucleotide polymorphism STK15 F31I as a low-penetrance risk allele for breast cancer, but its prevalence and risk association in the Brazilian population have not been determined. The goal of this study was to identify the frequency of this polymorphism in the Brazilian setting. Considering the high degree of admixture of our population, it is of fundamental importance to validate the results already reported in the literature and also to verify the relationship between this variant and breast cancer risk. A total of 750 women without breast cancer were genotyped using the TaqMan PCR assay for STK15 F31I polymorphism. Clinical information was obtained from review of the medical records and mammographic density from the images obtained using the BI-RADS System. The estimated risk of developing cancer was calculated according to the Gail model. The genotypic frequencies observed in this study were 4.5, 38.7, and 56.6%, respectively, for the STK15 F31I AA, AT and TT genotypes. The AT and AA genotypes were encountered significantly more often in premenopausal women with moderately dense, dense and heterogeneously dense breast tissue (P = 0.023). In addition, the presence of the TT genotype was significantly associated with age at menarche ≥12 years (P = 0.023). High mammographic density, associated with increased breast cancer risk, was encountered more frequently in premenopausal women with the risk genotypes STK15 F31I AA and AT. The genotypic frequencies observed in our Brazilian sample were similar to those described in other predominantly European populations.

TP53 PIN3 and MDM2 SNP309 polymorphisms as genetic modifiers in the Li-Fraumeni syndrome: impact on age at first diagnosis.

BACKGROUND: Li-Fraumeni and Li-Fraumeni-like syndromes (LFS/LFL), characterised by the development of multiple early onset cancers with heterogeneous tumour patterns, are associated with germline TP53 mutations. Polymorphisms in the TP53 pathway (TP53 PEX4 at codon 72, rs1042522; MDM2 SNP309, rs2279744) have modifier effects on germline TP53 mutations that may account for the individual and familial diversity of tumour patterns. METHODS AND RESULTS: Four polymorphisms were analysed in a series of 135 Brazilian LFS/LFL cancer patients (32 TP53 mutation carriers and 103 wild-type subjects). We report for the first time that another polymorphism in the TP53 gene, TP53 PIN3 (rs17878362), has a strong modifier effect on germline TP53 mutations. This polymorphism, which consists of a 16 bp duplication in intron 3 (A1, non-duplicated allele; A2, duplicated allele), is associated with a difference of 19.0 years in the mean age at the first diagnosis in TP53 mutation carriers (n = 25, A1A1: 28.0 years; n = 7, A1A2: 47.0 years; p = 0.01). In addition, cancer occurrence before the age of 35 years is exclusively observed in A1A1 homozygotes. In this series, the effect of TP53 PEX4 and MDM2 SNP309 on age at diagnosis was similar to the one reported in other series and was smaller than the one of TP53 PIN3 (TP53 PIN3: difference of 19.0 years; TP53 PEX4: 8.3 years; MDM2 SNP309: 12.5 years). CONCLUSION: These results suggest that TP53 PIN3 is another polymorphism in the TP53 pathway that may have a modifier effect on germline TP53 mutations and may contribute to the phenotypic diversity of germline TP53 mutations associated with LFS/LFL patients.

Founder effect of the BRCA1 5382insC mutation in Brazilian patients with hereditary breast ovary cancer syndrome.

The 5382insC mutation in BRCA1 is a frequently reported mutation, being very prevalent in Central and Eastern Europe. This mutation was recurrently reported in Brazil and one case was reported Portugal, but not in Spain and other South-American countries,. We analyzed the haplotypic profile of seven Brazilian carriers of 5382insC to characterize a possible founder effect. The analyses indicated that mutation carriers shared an identical haplotype. The absence of this mutation in Spain, other South American countries, and sub-Saharan populations, as well as the patients' own ancestry, point to a significant Central or Eastern European contribution to the present genetic background of Brazilian population, different from the population structuring of remaining South American countries.

An alternative protocol for DNA extraction from formalin fixed and paraffin wax embedded tissue.

BACKGROUND: DNA extraction from paraffin wax embedded tissue requires special protocols, and most described methods report an amplification success rate of 60-80%. AIMS: To propose a simple and inexpensive protocol consisting of xylene/ethanol dewaxing, followed by a kit based extraction. METHOD: Xylene/ethanol dewaxing was followed by a long rehydration step and a kit based DNA extraction step. RESULTS: This method produced a 100% amplification success rate for fragments of 121 to 227 bp for tamponated formalin fixed paraffin wax embedded tissue. CONCLUSION: This cost effective and non-laborious protocol can successfully extract DNA from tamponated formalin fixed paraffin wax embedded tissue and should facilitate the molecular analysis of a large number of archival specimens in retrospective studies.

Fabry disease: twenty-two novel mutations in the alpha-galactosidase A gene and genotype/phenotype correlations in severely and mildly affected hemizygotes and heterozygotes.

BACKGROUND: Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-chromosomal gene encoding the lysosomal exoglycosidase, alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22). The nature of the molecular lesions in the alpha-Gal A gene in 36 unrelated families was determined in order to provide precise heterozygote detection, prenatal diagnosis, and to define genotype/phenotype correlations. METHODS: Genomic DNA was isolated from affected males and/or carrier females from 36 unrelated families with Fabry disease. The entire alpha-Gal A coding region and flanking intronic sequences were analyzed by PCR amplification and solid-phase or cycle sequencing. Markers closely linked to the alpha-Gal A gene were analyzed to determine if probands with the same mutations were related. RESULTS: Twenty-two novel mutations were identified including 10 missense (P40L, W95S, S148N, C172R, M187V, N224S, W226R, A230T, D266H, N320Y), three nonsense (Y134X, C142X, W204X in two families), three splice-site defects (IVS2(+1), IVS3(+1), IVS4(+1)) and six small deletions or insertions (26delA in two families, 672ins37, 774delAC, 833insA, 1139delC, 1188insT). Of the remaining 12 families (33.3%), each had a previously identified mutation, eight of which occurred at CpG dinucleotides including R112C (two families), R112H, R227Q, R227X (three families), and R301Q. Haplotype analysis of the mutant alleles that occurred in two or three presumably unrelated families revealed that the families with the rare novel alleles (W204X and 26delA) were probably related, whereas those with mutations involving CpG dinucleotides (R112C and R227X) were not, the latter being consistent with their origins as independent mutational events. Genotype/phenotype correlations revealed that certain mutations previously found in mild variant patients also were found in classic patients. In addition, the genotypes and spectrum of phenotypic severity were determined in five heterozygotes with no family history. CONCLUSIONS: These results illustrate the molecular heterogeneity of the lesions causing Fabry disease and emphasize the fact that CpG dinucleotides constitute important hot spots for mutation in the alpha-Gal A gene. These studies also permit precise heterozygote detection and prenatal diagnosis in these families, and delineate phenotype-genotype correlations in this disease.

Fabry disease: comparison of enzymatic, linkage, and mutation analysis for carrier detection in a family with a novel mutation (30delG).

Fabry disease (FD) is an X-linked recessive disorder caused by the deficient activity of the lysosomal enzyme alpha-galactosidase A (alpha-Gal A). Affected males are reliably diagnosed by demonstration of deficient alpha-Gal A activity in plasma or leukocytes. However, identification of female carriers is problematic due to Lyonization, requiring mutation identification and/or linkage studies for accurate carrier detection. Here, we describe a large Brazilian kindred with Fabry disease that permitted comparison of biochemical and molecular diagnostic techniques. Initially, the plasma alpha-Gal A activities were determined in at-risk affected males and potential female carriers; affected males were readily diagnosed, while the females had variable results. To detect carrier females, haplotype analysis using 10 polymorphic markers adjacent to the gene was performed. Subsequently, solid-phase direct sequencing of the alpha-Gal A gene demonstrated a novel single base deletion in exon 1 (30delG). Discrepancies were observed between the enzymatic and molecular diagnoses in two at-risk females. These findings emphasize the need for precise heterozygote diagnosis by mutation and/or haplotype analyses in all families with Fabry disease.

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.

Non-pseudogene-derived complex acid beta-glucosidase mutations causing mild type 1 and severe type 2 gaucher disease.

Gaucher disease is an autosomal recessive inborn error of glycosphingolipid metabolism caused by the deficient activity of the lysosomal hydrolase, acid beta-glucosidase. Three phenotypically distinct subtypes result from different acid beta-glucosidase mutations encoding enzymes with absent or low activity. A severe neonatal type 2 variant who presented with collodion skin, ichthyosis, and a rapid neurodegenerative course had two novel acid beta-glucosidase alleles: a complex, maternally derived allele, E326K+L444P, and a paternally inherited nonsense mutation, E233X. Because the only other non-pseudogene-derived complex allele, D140H+E326K, also had the E326K lesion and was reported in a mild type 1 patient with a D140H+E326K/K157Q genotype, these complex alleles and their individual mutations were expressed and characterized. Because the E233X mutation expressed no activity and the K157Q allele had approximately 1% normal specific activity based on cross-reacting immunologic material (CRIM SA) in the baculovirus system, the residual activity in both patients was primarily from their complex alleles. In the type 1 patient, the D140H+E326K allele was neuroprotective, encoding an enzyme with a catalytic efficiency similar to that of the N370S enzyme. In contrast, the E326K+L444P allele did not have sufficient activity to protect against the neurologic manifestations and, in combination with the inactive E233X lesion, resulted in the severe neonatal type 2 variant. Thus, characterization of these novel genotypes with non-pseudogene-derived complex mutations provided the pathogenic basis for their diverse phenotypes.

Say syndrome: a new case with cystic renal dysplasia in discordant monozygotic twins.

Say syndrome is a rare condition characterized by cleft palate, short stature, and microcephaly. Abu-Libdeh et al. [1993: Am J Med Genet 45:358-360] described a case with cystic renal dysplasia. We describe monozygotic twins discordant for the syndrome with kidney dysplasia. A postzygotic mutation is proposed as the cause of this autosomal dominant syndrome in this case.

Prenatal diagnosis of a familial interchromosomal insertion of Y chromosome heterochromatin.

An apparently unbalanced karyotype containing an abnormal chromosome 11 was identified in a 16-week female fetus by analysis of cultured amniocytes. Fluorescence in situ hybridization (FISH) with a chromosome 11 paint identified the presence of an insertion in band 11q24. Parental karyotyping documented an unbalanced karyotype with the same der(11) chromosome in the phenotypically normal father. CBG-banding and FISH identified the insertion to be Yq12 heterochromatin: 46,XY, der(11)ins(11;Y)(q24;q12q12).ish der(11) (wcp11+,DYZ1+). The same der(11) chromosome was also found in the phenotypically normal paternal grandmother, demonstrating this additional Y chromosomal material did not affect normal female sexual development or fertility. The parents elected to continue the pregnancy and a normal girl was born at term, further confirming that this rare familial variant has no clinical significance. This case illustrates the importance of family studies, appropriate banding, and FISH analyses to accurately characterize apparent chromosomal abnormalities.

Thalidomide, a current teratogen in South America.

Thalidomide, mainly used for the treatment of leprosy, is a current teratogen in South America, and it is reasonable to assume that at present this situation is affecting many births in underdeveloped countries. Moreover, the potential re-marketing of thalidomide for the treatment of a large variety of diseases may extend the problem to the developed world. When the drug is available, the control of its intake during early pregnancy is very difficult since most pregnancies are unintended. The ongoing occurrence of thalidomide embryopathy cases went undetected by the ECLAMC, due to several factors: (1) low populational coverage through this monitoring system; (2) pre-existence of the teratogen with its effects present in both baseline (expected) and monitored (observed) materials; and (3) lack of a defined phenotype to be monitored. Thus, if thalidomide re-enters the market throughout the world, due to the wide range of new applications, occurrence of phocomelia alone might not be sufficient to detect its effects. By a case-reference approach, the ECLAMC registered 34 thalidomide embryopathy cases born in South America after 1965 whose birthplaces correspond to endemic areas for leprosy. Phocomelia was found in five of eleven fully described cases. Thus, phocomelia alone is neither specific nor sufficient to serve as a suitable phenotype to survey the teratogenic effects of thalidomide. Therefore, a thalidomide-like phenotype, defined as any bilateral upper and/or lower limb reduction defect of the preaxial and/or phocomelia types, should be included in the routine surveillance of birth defects in all programmes.

Maternal hyperphenylalaninaemia as a cause of microcephaly and mental retardation.

We attempted to evaluate the role of maternal hyperphenylalaninaemia (HPA) as an isolated cause of mental retardation and microcephaly in children. This transversal study observed the plasma phenylalanine from mothers of 161 children with mental retardation and/or microcephaly of unknown origin. In this sample, we found two women with previously undiagnosed HPA, a frequency (2/161) higher than expected for our general population (1:12 500) (p < 0.001). We concluded that the plasma phenylalanine levels should be determined during preconceptional evaluation of every woman of reproductive age that already has had a child affected either by mental retardation or microcephaly of unknown cause. It is particularly significant where women currently having their pregnancies have not been screened for phenylketonuria as newborns, as happens in most developing countries.