Connexin-26 gene analysis in hearing-impaired newborns

The efficacy and utility of the Connexin-26 (Cx-26) gene (also called GJB2) analysis from DNA isolated from Guthrie newborn screening cards is demonstrated. This analysis precisely defined a major cause of prelingual nonsyndromic deafness in those children requiring amplification in our study. Guthrie cards were obtained from 49 deaf children requiring amplification identified over the last 5 years by the Rhode Island Newborn Screening Program. Children with syndromes or other recognizable causes of hearing loss were excluded. DNA was extracted from the Guthrie cards and analyzed sequentially for the Cx-26 35delG mutation and then for the 167delT mutation followed by gene sequencing on remaining heterozygotes. Three of 42 children were 35delG homozygotes; 2/42 children were 35delG/167delT compound heterozygotes. One child was identified as being a 35delG heterozygote with no other mutation found by sequencing. Nine Guthrie cards yielded no amplification or uninterpretable results. Cx-26 mutations were identified as causing 11.9% of the deafness in the children studied. In conclusion, Cx-26 analysis is an important test that identifies a major cause of prelingual nonsyndromic deafness. Molecular analysis of hearing-impaired newborns will be important for genetic counseling in these families. Failures with Guthrie cards may make use of other collection methods preferable.     

Phenylketonuria in Iranian population: a study in institutions for mentally retarded in Isfahan

Phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene (12q22-q24) resulting in a primary deficiency of the PAH enzyme activity, intolerance to the dietary intake of phenylalanine (Phe) and production of the phenylketonuria (PKU) disease. To date there have been no reports on the molecular analysis of PKU in Iranian population. In this study, the states of the PKU disease in terms of prevalence and mutation spectrum among patients reside in the institutions for mentally retarded in Isfahan was investigated. In the first step, 611 out of 1541 patients with PKU phenotype or severe mental retardation were screened for the PKU disease using the Guthrie bacterial inhibition assay (GBIA) followed by HPLC. Among the patients screened 34 (5.56%) were found positive with abnormal serum Phe of above 7mg/dl. In the next step, the presence of 18 common mutations of the PAH gene in 26 of the patients with classical PKU (serum Phe above 20mg/dl) was investigated, using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Of the 52 independent mutant alleles that were analyzed, 34 (65.38%) were genotyped showing 8 mutations as follows: R252W (15.38%), Q232Q (13.46%), R261Q (7.69%), delL364 (7.69%), IVS10-11g>a (5.77%), L333F (5.77%), V245V (5.77%) and S67P (3.85%). The results from this study may serve as a reference to analyze the PKU mutations in other part of Iran, and to establish diagnostic tests for carrier detection and prenatal diagnosis of the PKU disease in Iranian population.     

Genetic analysis carried out on blood-spots of phenylalanine hydroxylase-deficient newborns detected by northeastern Italian neonatal screening

The aim of this work was to perform genetic analysis on 18 different blood-spot samples collected from neonates detected as hyperphenylalaninemic by Northeastern Italian screening program. DNA was extracted from blood-spots. Exons/introns of PAH gene were amplified by polymerase chain reaction (PCR), and PCR products were purified and sequenced with both forward and reverse primers. The most frequent mutations were IVS12nt1g>a (16.7%) and R408W, P281L and L48S (all together 11.1%). As expected, compound heterozygosity was the usual finding; homozygosity was found only in two patients with R158Q and IVS2nt5g>c mutations. The V230I mutation was reported for the first time in Italy. We found six previously described polymorphisms (V245V, IVS4nt47c>t, IVS2nt19t>c, IVS3nt-22c>t, IVS5nt-54a>g, and E280>Q280). To our knowledge, four genotypes were not previously described: R158Q/V230I present in one patient with classical PKU; and L48S/R408Q, A403V/IVS2nt-13t>g, and G272X/V230I present in patients showing HPA phenotype. Most of the mutations were located in the exons 12 and 7 and in exon/intron 2 (83.3% detection of total mutations in PKU or HPA patients of Northeastern Italy). From a practical viewpoint, the genetic analysis of blood-spots collected on Guthrie cards for neonatal screening for PKU could be a simple method to establish the genotype of neonates. Consequently, the genotype/phenotype correlation could lead to a more accurate diagnosis and prognosis for families.     

Neonatal detection of the 35delG mutation of the GJB2 gene in families at risk for deafness

About half of congenitally deaf children that have a recessively inherited sensorineural deafness are born from normal-hearing parents and have no risk factor for hearing loss. Mutation 35delG in the connexin-26 gene is in European populations the basis for around half of all recessively inherited prelingual sensorineural deafness. The aim of our study was to assess the efficacy and utility of the 35delG mutation of the connexin-26 gene analysis for neonates at familial risk, from DNA isolated from Guthrie newborn screening cards. Newborns who had consanguineous parent and/or a familial history of deafness underwent connexin-26 gene analysis from DNA isolated from Guthrie cards and two hearing screening tests (transient evoked otoacoustic emissions, and auditory brainstem recordings). 24 newborns were includes in this pilot study; one of them is homozygous for the 35delG mutation and had abnormal hearing screening tests; all the others newborns had normal connexin gene and at least one normal hearing screening test. Detection on connexin-26 gene mutation is feasible in selected at-risk newborns on one additional blood spot on Guthrie card.     

Recurrent mutation in the human phenylalanine hydroxylase gene.

We report the identification of a missense mutation of Glu280 to Lys280 in the phenylalanine hydroxylase (PAH) gene of a phenylketonuria (PKU) patient in Denmark. The mutation is associated with haplotype 1 of the PAH gene in this population. This mutation has previously been found in North Africa, where it is in linkage disequilibrium with haplotype 38. While it is conceivable that this mutation could have been transferred from one haplotype background to another by a double crossover or gene conversion event, the fact that the mutation is exclusively associated with the two different haplotypes in the two distinct populations supports the hypothesis that these two PKU alleles are the result of recurrent mutations in the human PAH gene. Furthermore, since the site of mutation involves a CpG dinucleotide, they may represent hot spots for mutation in the human PAH locus.     

Whole genome amplification on DNA from filter paper blood spot samples: an evaluation of selected systems

As the number of single-nucleotide polymorphism (SNP) screening and other mutation scanning studies have increased explosively, following the development of high-throughput instrumentation, it becomes even more important to have sufficient template DNA. The source of DNA is often limited, especially in epidemiological studies, which require many samples as well as enough DNA to perform numerous SNP screenings or mutation scannings. Therefore, the aim is to solve the problem of stock DNA limitation. This need has been an important reason for the development of whole genome amplification (WGA) methods. Several systems are based on Phi29 polymerase multiple displacement amplification (MDA) or on DNA fragmentation (OmniPlex). Using TaqMan SNP genotyping assays, we have tested four WGA systems -- AmpliQ Genomic Amplifier Kit, GenomiPhi, Repli-g, and GenomePlex -- on DNA extracted from Guthrie cards to evaluate the amplification bias, concordance- and call rates, cost efficiency, and flexibility. All systems successfully amplified picograms of DNA from Guthrie cards to micrograms of product without loss of heterozygosity and with minimal allelic bias. A modified AmpliQ set up was chosen for further evaluation. In all, 2,000 SNP genotyping results from amplified and nonamplified samples were compared and the concordance rates between the samples were 99.7%. The call rate using the TaqMan system was 99.8%. DNA extracted from Guthrie cards and amplified with one of the four evaluated WGA systems is applicable in epidemiological genetic screenings. System choice should be based on requirements for system flexibility, product yield, and use in subsequent analysis.     

The promise and challenges of blood spot methylomics

Epigenome-wide association studies (EWAS) are being extensively performed to identify epigenetic variants associated to complex diseases. However, EWAS may identify variants that are disease-induced rather than disease-causal. Recent studies have highlighted the use of Guthrie cards to profile the methylome at birth, permitting researchers to find epigenetic variants present in patients before they are diagnosed with clinical disease, with the implicit suggestion that these variants are more likely to be disease causal. The use of Guthrie cards for research purposes throws up a number of ethical issues. We review here the promises and pitfalls of Guthrie cards for disease research.     

Stored Guthrie cards as DNA "banks".

Recently there has been much discussion about the possibility of using dried blood spots on Guthrie cards as a source of DNA for research or testing purposes. The collections of Guthrie cards stored by state newborn-screening laboratories can thus be viewed as inchoate "DNA banks." This has generated concern among some persons who are interested in preserving the privacy of medical records. This study examines the policies of state newborn-screening laboratories in the United States, regarding their retention of Guthrie cards and the degree to which they permit the sharing of those cards with various third parties. We found that although most laboratories retain their cards, if at all, for only a short time, a growing number plan to keep them for an extended period--and, in several cases, indefinitely. We also found that although most laboratories would decline to release individually identifiable blood spots from the cards to third parties without a written release or other explicit authorization, a large number would at least consider sharing anonymous cards for research purposes.     

Regional distribution of heterozygous 657del5 mutation carriers of theNBS1 gene in Wielkopolska province (Poland)

The homozygous 657del5 mutation, called Slavic mutation, of the NBS1 gene, causes the Nijmegen Breakage Syndrome (NBS). This syndrome is connected with a high incidence of malignancies in early childhood. A high frequency of NBS heterozygotes was found among patients with melanoma, breast, ovary and prostate cancer. The aim of our research was to determine the frequency of 657del5 mutation of the NBS1 gene in the population of Wielkopolska province. For this purpose, we analysed blood samples from anonymous Guthrie cards. In a group of 2090 newborns from the whole province, we found 16 heterozygous mutation carriers. The frequency of 1/131 is higher than 1/190 reported for populations from other regions in Poland. We observed differential regional distribution of heterozygous 657del5 mutation carriers within the province: among 464 samples from the eastern part of Wielkopolska we found 6 carriers (1/77), in contrast to the southern part without any carrier among 625 samples analysed. The high mean frequency of heterozygous 657del5 mutation (1/131) in Wielkopolska province may be associated with cancer incidence in this region.     

Phenylketonuria-the guthrie screening test-a method of quantitation, observations on reliability and suggestions for improvement

The Guthrie bacterial inhibition assay method of screening neonatal infants for phenylketonuria (pku) initiated mass screening for inborn errors of metabolism. It is a simple, cheap procedure admirably suited to private local use as against private or public central use. However, using parallel fluorometric determinations as a basis for comparison, and 4 mg per 100 ml serum phenylalanine as a presumptive positive threshhold, the Guthrie test yielded 53 per cent "false negatives." Extrapolating from a combination of our data and reported phenylalanine levels at three days of age or less in proved pku patients, it is estimated the Guthrie test might fail to detect one of 25 pku patients screened at three days of age or less. Means to diminish this risk are considered.     

Retrospective study of the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Guthrie cards from a large cohort of neonatal screening for cystic fibrosis

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-activated chloride channel, and in individuals with both alleles of the gene mutated, symptoms of CF disease are manifest. With more than 300 mutations so far described in the gene the profile of mutant alleles in a population is specific to its ethnic origin. For an analysis with an unbiased recruitment of the CF alleles in neonates of similar origin (Normandy, France), we have retrospectively analyzed the Guthrie cards of affected newborns, diagnosed by the immunoreactive trypsinogen (IRT) assay. Analysis of the 27 exons of the CFTR gene using a GC clamp denaturing gradient gel electrophoresis (DGGE) assay has enabled us to identify over 96% of the mutated alleles. Two of these were novel mutations. We would like to propose this strategy as an efficient method of retrospective molecular genetic diagnosis that can be performed wherever Guthrie cards can be obtained. Knowledge of rare alleles could be a prerequisite for CF therapy in the future.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

Molecular basis for nonphenylketonuria hyperphenylalaninemia.

Nonphenylketonuria hyperphenylalaninemia (non-PKU HPA) is defined as phenylalanine hydroxylase (PAH) deficiency with blood phenylalanine levels below 600 mumol/liter (i.e., within the therapeutic range) on a normal dietary intake. Haplotype analysis at the PAH locus was performed in 17 Danish families with non-PKU HPA, revealing compound heterozygosity in all individuals. By allele-specific oligonucleotide (ASO) probing for common PKU mutations we found 12 of 17 non-PKU HPA children with a PKU allele on one chromosome. To identify molecular lesions in the second allele, individual exons were amplified by polymerase chain reaction and screened for mutations by single-strand conformation polymorphism. Two new missense mutations were identified. Three children had inherited a G-to-A transition at codon 415 in exon 12 of the PAH gene, resulting in the substitution of asparagine for aspartate, whereas one child possessed an A-to-G transition at codon 306 in exon 9, causing the replacement of an isoleucine by a valine in the enzyme. It is further demonstrated that the identified mutations have less impact on the heterozygote's ability to hydroxylate phenylalanine to tyrosine compared to the parents carrying a PKU mutation. The combined effect on PAH activity explains the non-PKU HPA phenotype of the child. The present observations that PKU mutations in combination with other mutations result in the non-PKU HPA phenotype and that particular mutation-restriction fragment length polymorphism haplotype combinations are associated with this phenotype offer the possibility of distinguishing PKU patients from non-PKU individuals by means of molecular analysis of the hyperphenylalaninemic neonate and, consequently, of determining whether a newborn child requires dietary treatment.     

RFLP haplotyping and mutation analysis of the phenylalanine hydroxylase gene in Dutch phenylketonuria families

Restriction fragment length polymorphism haplotyping of mutated and normal phenylalanine hydroxylase (PAH) alleles in 49 Dutch phenylketonuria (PKU) families was performed. All mutant PAH chromosomes identified by haplotyping (n = 98) were screened for eight of the most predominant mutations. Compound heterozygosity was proven in 40 kindreds. Homozygosity was found for the IVS12nt1 mutation in 5 families, and for the R158Q and IVS10nt546 mutations in one family each. All patients from these families suffer from severe PKU, providing additional proof that these mutations are deleterious for the PAH gene. Genotypical heterogeneity was evident for mutant haplotype 1 (n = 27) carrying the mutations R261Q (n = 12), E280K (n = 4), P281L (n = 1) and unknown (n = 10), and likewise for mutant haplotype 4 (n = 30) carrying the mutations R158Q (n = 13), Y414C (n = 1) and unknown (n = 16). Mutant haplotype 3 (n = 20), in tight association with mutation IVS12nt1, appeared to be in strong linkage disequilibrium (LDE) with its normal counterpart allele (n = 4). Mutant haplotype 6 (n = 4), in tight association with the IVS10nt546 mutation, showed moderate LDE with its counterpart allele (n = I). The distribution of the mutant PAH haplotypes 1, 3 and 4 among the Dutch PKU population resembles that in other Northern and Western European countries, but it is striking that mutant haplotype 2 and its associated mutation R408W is nearly absent in The Netherlands, in strong contrast to its neighbouring countries.     

Aberrant splicing of phenylalanine hydroxylase mRNA: the major cause for phenylketonuria in parts of southern Europe.

We report a mutation within the phenylalanine hydroxylase (PAH) gene that causes aberrant splicing of the mRNA and that is in tight association with chromosomal haplotypes 6, 10, and 36. Because of the high frequency of these particular haplotypes in Bulgaria, Italy, and Turkey, it appears to be one of the more frequent defects in the PAH gene causing classical phenylketonuria in this part of Europe. The mutation is a G to A transition at position 546 in intron 10 of the PAH gene, 11 bp upstream from the intron 10/exon 11 boundary. It activates a cryptic splice site and results in an in-frame insertion of 9 nucleotides between exon 10 and exon 11 of the processed mRNA. Normal amounts of liver PAH protein are present in homozygous patients, but no catalytic activity can be detected. This loss of enzyme activity is probably caused by conformational changes resulting from the insertion of three additional amino acids (Gly-Leu-Gln) between the normal sequences encoded by exon 10 and exon 11.     

Prevalence of HIV antibody and pregnancy in Tayside, 1984-9: background to screening.

OBJECTIVE--To determine age specific prevalence of HIV antibody, incidence of pregnancy, and likelihood of detection and correct assignment to risk category by antenatal screening of women known to be positive for HIV antibody, from 1984 to 1989. DESIGN--Retrospective analysis of reproductive history and risk behaviour of women positive for HIV antibody and prediction of detection by screening on the basis of blood group samples, Guthrie tests, and rubella tests. SETTING--City of Dundee, where the prevalence of HIV is high, since the appearance of HIV in 1984, predominantly among heterosexual intravenous drug users. PATIENTS--All (61) women known to be positive for HIV antibody who had had clinically indicated tests, for whom case notes were available for 60. MAIN OUTCOME MEASURES--Risk assessment according to case notes and reported to the laboratory, incidence of infection, geographical location, age, date of positive test result, and reproductive history. RESULTS--With 61 infected women the overall minimum prevalence among women within the city of Dundee was 0.67/1000 and 2.9/1000 among women in their third decade. Of the 60 women whose reproductive history was available, 35 had 57 pregnancies, 36 of which occurred after seroconversion was known to have taken place, representing 8.7% of the total number of affected pregnancies reported for the United Kingdom. If antenatal screening for HIV had been performed between 1984 and 1989 it could not have detected positivity for HIV antibody in 25 (42%) women who had no pregnancies during this time. Among the remaining 35 women, screening samples taken for blood grouping could have identified a maximum of 34 (57%), samples taken to check rubella susceptibility a maximum of 22 (37%), and blood spots on Guthrie cards a maximum of 19 (32%). Retesting would have occurred in 14 women 33 times with samples taken for blood grouping, but three and four women would have been tested twice using samples taken for rubella testing and Guthrie cards respectively. Anonymous screening would have been unable to determine risk category as a history of intravenous drug use was known in 47 (79%) women before testing but this was increased by a further 5 (8%) who admitted to it after the test result was known. CONCLUSION--Interpreting the results of antenatal screening programmes will be complex and will underestimate overall prevalence of HIV antibody among women; this will be exaggerated by strategies based on anonymous testing with Guthrie cards or on samples taken for rubella testing, which do not include women who have had an earlier loss of pregnancy. Only open testing with consent will permit satisfactory attribution to     

Phenylketonuria—The Guthrie Screening Test—A Method of Quantitation,Observations on Reliability and Suggestions for Improvement

The Guthrie bacterial inhibition assay method of screening neonatal infants for phenylketonuria (pku) initiated mass screening for inborn errors of metabolism. It is a simple, cheap procedure admirably suited to private local use as against private or public central use. However, using parallel fluorometric determinations as a basis for comparison, and 4 mg per 100 ml serum phenylalanine as a presumptive positive threshhold, the Guthrie test yielded 53 per cent “false negatives.” Extrapolating from a combination of our data and reported phenylalanine levels at three days of age or less in proved pku patients, it is estimated the Guthrie test might fail to detect one of 25 pku patients screened at three days of age or less. Means to diminish this risk are considered.     

Decrease of 3243 A-->G mtDNA mutation from blood in MELAS syndrome: a longitudinal study

It is widely held that changes in the distribution of mutant mtDNAs underlie the progressive nature of mtDNA diseases, but there are few data documenting such changes. We compared the levels of 3243 A-->G mutant mtDNA in blood at birth from Guthrie cards and at the time of diagnosis in a blood DNA sample from patients with mitochondrial encephalopathy, lactic acidosis, and strokelike episodes (MELAS) syndrome. Paired blood DNA samples separated by 9-19 years were obtained from six patients with MELAS. Quantification of mutant load, by means of a solid-phase minisequencing technique, demonstrated a decline (range 12%-29%) in the proportion of mutant mtDNA in all cases (P=.0015, paired t-test). These results suggest that mutant mtDNA is slowly selected from rapidly dividing blood cells in MELAS.     

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.     

A newborn screening method for cerebrotendinous xanthomatosis using bile alcohol glucuronides and metabolite ratios

Cerebrotendinous xanthomatosis (CTX) is a treatable neurodegenerative metabolic disorder of bile acid synthesis in which symptoms can be prevented if treatment with chenodeoxycholic acid supplementation is initiated early in life, making CTX an excellent candidate for newborn screening. We developed a new dried blood spot (DBS) screening assay for this disorder on the basis of different ratios between the accumulating cholestanetetrol glucuronide (tetrol) and specific bile acids/bile acid intermediates, without the need for derivatization. A quarter-inch DBS punch was extracted with methanol, internal standards were added, and after concentration the extract was injected into the tandem mass spectrometer using a 2 min flow injection analysis for which specific transitions were measured for cholestanetetrol glucuronide, taurochenodeoxycholic acid (t-CDCA), and taurotrihydroxycholestanoic acid (t-THCA). A proof-of-principle experiment was performed using 217 Guthrie cards from healthy term/preterm newborns, CTX patients, and Zellweger patients. Using two calculated biomarkers, tetrol:t-CDCA and t-THCA:tetrol, this straightforward method achieved an excellent separation between DBSs of CTX patients and those of controls, Zellweger patients, and newborns with cholestasis. The results of this small pilot study indicate that the tetrol:t-CDCA ratio is an excellent derived biomarker for CTX that has the potential to be used in neonatal screening programs.