Analysis of the phenylalanine hydroxylase gene in the Spanish population: mutation profile and association with intragenic polymorphic markers.

The aim of this study was to characterize the phenylketonuria (PKU) alleles in the Spanish population, by both identifying the causative mutations and analyzing the RFLP haplotypes and the VNTR and short-tandem-repeat alleles associated with the phenylalanine hydroxylase (PAH) gene. We have investigated 129 independent mutant chromosomes, using denaturing gradient gel electrophoresis (DGGE) and direct sequencing. Ninety percent of the alleles were identified, and a total of 40 different mutations were detected. The mutational spectrum includes seven previously unreported mutations: P122Q, D129G, P147S, D151G, A165T, S196fs, and P407S. Seven mutations represent 43% of the Spanish PKU alleles, the most common being IVS10nt-11g-->a (14.7%), I65T (8.5%), and V388M (6.2%). The remaining 33 mutations are rare. The mutation profile and relative frequencies are markedly different from those in northern Europe, also showing unique features compared with those in other, southern European populations. The association analysis with polymorphic markers in the PAH gene provides valuable information for population-genetic studies and investigation of the origins of the mutations. This study may serve as reference in the analysis of the contemporary distributions and frequencies of the PKU mutations in related populations, with particular relevance in Latin American countries.     

Phenylketonuria in Spain: RFLP haplotypes and linked mutations

In order to investigate the molecular basis of phenylketonuria (PKU) in Spain, we analyzed the restriction fragment length polymorphism (RFLP) haplotypes and common mutations in the phenylalanine hydroxylase (PAH) gene in 32 unrelated Spanish PKU families. The distribution of RFLP haplotypes differs from that of northern Europe. Mutant haplotypes 2 and 3 were completely absent in our sample. Approximately 65% of the mutant alleles are confined to three RFLP haplotypes, namely haplotypes 1, 6 and 9, also frequently found in other Mediterranean populations. We screened for previously described PKU mutations using the polymerase chain reaction and allele-specific oligonucleotides, and found IVS10,165T, E280K and P281L as the major mutations, representing 41% of the PKU alleles. Other mutations found were Y414C, and a new one, P244L. Mutations R408W and IVS12, prevalent in northern Europe, as well as others present in southern European populations (R252W, R261Q, L249F) were not detected in our sample. Our results reveal the genetic heterogeneity present in the Spanish PKU population, which shows similarities to others of Mediterranean origin.     

Phenylketonuria mutations and their relation to RFLP haplotypes at the PAH locus in Czech PKU families

A detailed study of the mutant phenylalanine hydroxylase (PAH) gene from the eastern part of the Czech Republic (Moravia) is reported. A total of 190 mutant alleles from 95 phenylketonuria (PKU) families were analyzed for 21 prevalent Caucasian mutations and restriction fragment length polymorphism /variable number of tandem repeats (RFLP/VNTR) haplotypes. Eighty per cent of all mutant alleles were found to carry 11 mutations. The most common molecular defect was the mutation R408W (55.3%), with a very high degree of homozygosity (34.6%). Each of four other mutations (R158Q, R243X, G272X, IVS12nt1) accounted for more than 3% of PKU alleles. Rarely present were mutations IVS10nt546 (2.6%), R252W (2.6%), L48S (2.1%), R261Q (1.6%), Y414C (1.0%) and I65T (0.5%). Mutations that have been predominantly described in southern Europe (IVS7nt1, A259V, Y277D, R241H, T278N) were not detected. A total of 14 different mutant haplotypes were observed. Three unusual genotype-haplotype associations were identified (R158Q on haplotypes 2.3 and 7.8 and R252W on haplotype 69.3). There was a strong association between the mutation R408W and haplotype 2.3 (54.7%). Heterogeneity was found at mutations R408W (haplotypes 2.3 and 5.9), R158Q (haplotypes 4.3, 2.3 and 7.8) and IVS10nt546 (haplotypes 6.7 and 34.7). The molecular basis of PKU in the Moravian area appears to be relatively homogeneous in comparison with other southern and western European populations, thus providing a good starting point for prenatal diagnosis and early clinical classification.     

Molecular characterization of phenylketonuria in Japanese patients

We characterized phenylalanine hydroxylase (PAH) genotypes of Japanese patients with phenylketonuria (PKU) and hyperphenylalaninemia (HPA). PKU and HPA mutations in 41 Japanese patients were identified by denaturing gradient gel electrophoresis and direct sequencing, followed by restriction fragment length polymorphism analysis to find a large deletion involving exons 5 and 6. Of 82 mutant alleles, 76 (92%) were genotyped showing 21 mutations. The major mutations were R413P (30.5%), R243Q (7.3%), R241 C (7.3%), IVS4nt-1 (7.3%), T278I (7.3%), E6nt-96A→g (6.1%), Y356X (4.9%), R111X (3.7%), and 442–706delE5/6 (2.4%). Eight new mutations (L52 S, delS70, S70P, Y77X, IVS3nt-1, A132 V, W187 C, and C265Y) and a polymorphism of IVS10nt-14 were detected. In vitro PAH activities of mutant PAH cDNA constructs were determined by a COS cell expression system. Six mutations, viz., R408Q, L52 S, R241 C, S70P, V388 M, and R243Q, had 55%, 27%, 25%, 20%, 16% and 10% of the in vitro PAH activity of normal constructs, respectively. The mean pretreatment phenylalanine concentration (0.83±0.21 mmol/l) of patients carrying the R408Q, R241 C, or L52 S mutation and a null mutation was significantly lower (P<0.0005) than that (1.99±0.65 mmol/l) of patients with both alleles carrying mutations associated with a severe genotype. Simple linear regression analysis showed a correlation between pretreatment phenylalanine concentrations and predicted PAH activity in 29 Japanese PKU patients (y=31.9–1.03x, r=0.59, P<0.0001). Genotype determination is useful in the prediction of biochemical and clinical phenotypes in PKU and can be of particular help in managing patients with this disorder. Received: 24 July 1998 / Accepted: 12 September 1998     

Rapid mutation screening of phenylketonuria by polymerase chain reaction-linked restriction enzyme assay and direct sequence of the phenylalanine hydroxylase gene: clinical application in northern Japan and northern China

We describe a simple and technically feasible method for mutation screening of the phenylalanine hydroxylase (PAH) gene and its application to Japanese and Chinese patients with hyperphenylalaninemia. The strategy is based on the identification of a nucleotide substitution by restriction enzyme analysis, coupled with PCR and direct sequencing of exon 7 of the PAH gene. Because the detection of various mutations can proceed simultaneously using the same technique, it is quite rapid and reproducible, making it possible to perform effective molecular diagnosis and carrier screening in most laboratories. Using this procedure, we found that the most common molecular defects were R413P in Hokkaido, Japan (35 %) and R243Q in Heilongjiang, China (50%). R111X, IVS4nt-1, and five mutations in exon 7 (R241C, R243Q, R252W, A259T, and S273P) accounted for 55% of phenylketonuria (PKU) alleles in Hokkaido. In Heilongjiang, the R111X, Y356X, and R408W mutations accounted for 35% of PKU alleles. Clinically, homozygotes or compound heterozygotes of null alleles, which express nonfunctional enzyme activity, were all associated with classic PKU. On the other hand, patients heterozygous for the R241C allele had a benign phenotype of mild hyperphenylalaninemia. The DNA diagnosis in early infancy can predict various PKU phenotypes, and can prove useful in decision-making concerning dietary therapy.     

Mutation in the structure of exon 7 of the phenylalanine hydroxylase in phenylketonuria patients from the Novosibirsk area

The spectrum and frequency of mutations of exon 7 of the gene for phenylalanine hydroxylase (PAH) were studied in 34 phenylketonuria (PKU) patients living in Novosibirsk oblast. The five most prevalent mutations constituted 17.64% of defective alleles: R243Q (1.47%), R252W (1.47%), R261Q (5.88%), E280K (1.47%), and P281L (7.35%). A neutral polymorphic locus V245V was found within exon 7.     

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.     

Frequencies of phenylalanine hydroxylase mutations I65T, R252W, R261Q, R261X, IVS10nt11, V388M, R408W, Y414C, and IVS12nt1 in Minas Gerais, Brazil

In order to determine the phenylketonuria (PKU) mutation spectrum in the population of Minas Gerais State, Brazil, 78 unrelated PKU patients found by the neonatal screening program from 1993 to 2003 were tested for nine phenylalanine hydroxylase mutations. These mutations were selected due to their high frequencies in other Brazilian populations and in Portugal, where the largest contingent of the Caucasian component of the Brazilian population originated from. The most frequent mutations were V388M (21%), R261Q (16%), IVS10nt11 (13.4%), I65T (5.7%), and R252W (5%). The frequencies of the other four mutations (R261X, R408W, Y414C, and IVS12nt1) did not reach 2%. By testing these nine mutations, we were able to identify 64% of the PKU alleles in our sample. V388M frequency was higher than in any other known population and almost three times larger than that observed in Portugal, probably reflecting genetic drift. The mutation profile, as well as the relative frequency of the different mutations, suggest that the Minas Gerais population more closely resembles that of Portugal than do the other Brazilian populations that have already been tested.     

Association between minihaplotypes and mutations at the PAH locus in Polish hyperphenylalaninemic patients

Hereditary hyperphenylalaninemia (HPA) is a disorder of amino acid metabolism and results from an insufficiency of hepatic phenylalanine hydroxylase (PAH). HPA phenotypes form a spectrum ranging from classical phenylketonuria (PKU) to mild hyperphenylalaninemia (MHP). The phenotypic diversity reflects heterogeneity at the molecular level, and more than 320 different mutations in the PAH gene are known to date. The association of 3 mutations (R408W, IVS10 and A403V) common in different European populations with a variable number tandem repeat (VNTR) and short tandem repeat sites (minihaplotype) in the PAH gene was examined in a group of Polish PKU and MHP patients. Additionally, minihaplotypes were established for another 16 mutations. The presented data support the hypothesis that the R408W/VNTR3/STR238 allele originated among pre-Indo-Europeans on the territory in present-day Lithuania and Belarus. Mutation IVS10nt-11g-->a (IVS10) is strongly associated with VNTR7/STR250 minihaplotype and is possibly of Mediterranean origin.     

Phenylalanine hydroxylase gene mutations in the United States: report from the Maternal PKU Collaborative Study.

The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g-->a, and Y414C, accounting for 18.7%, 7.8%, and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies < or = 1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment of mutations has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency.     

Mutations of the phenylalanine hydroxylase gene in patients with phenylketonuria in Shanxi,China

The variation in mutations in exons 3, 6, 7, 11 and 12 of the phenylalanine hydroxylase (PAH) gene was investigated in 59 children with phenylketonuria (PKU) and 100 normal children. Three single nucleotide polymorphisms were detected by sequence analysis. The mutational frequencies of cDNA 696, cDNA 735 and cDNA 1155 in patients were 96.2%, 76.1% and 7.6%, respectively, whereas in healthy children the corresponding frequencies were 97.0%, 77.3% and 8.3%. In addition, 81 mutations accounted for 61.0% of the mutant alleles. R111X, H64 > TfsX9 and S70 del accounted for 5.1%, 0.8% and 0.8% mutation of alleles in exon 3, whereas EX6-96A > G accounted for 10.2% mutation of alleles in exon 6. R243Q had the highest incidence in exon 7 (12.7%), followed by Ivs7 + 2 T > A (5.1%) and T278I (2.5%). G247V, R252Q, L255S, R261Q and E280K accounted for 0.8% while Y356X and V399V accounted for 5.9% and 5.1%, respectively, in exon 11. R413P and A434D accounted for 5.9% and 2.5%, respectively, in exon 12. Seventy-two variant alleles accounted for the 16 mutations observed here. The mutation characteristics and distributions demonstrated that EX6-96A > G and R243Q were the hot regions for mutations in the PAH gene in Shanxi patients with PKU.     

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.     

苯丙氨酸羟化酶（PAH）基因外显子7及其两侧内含子的突变研究

为探讨中国苯丙酮尿症（PKU）人群中苯丙氨酸羟化酶（PAH）基因外显子7的突变特征,对147例PKU患儿的294个PAH基因外显子7以及两侧部分内含子序列,应用PCR－单链构象多态性（SSCP）分析及基因序列分析的方法进行了筛查和确定。共发现13种突变基因：G239D、R241C、R241fs、R243Q、G247S、G247V、R252Q、L255S、R261Q、M276K、E280G、P281L、Ivs7+2T>A,其中7 种突变基因在中国PKU人群首次发现：G239D 、R241fs 、G247S 、E280G、L255S、R261Q、P281L,前4种在国际上尚未见到报道,并已提交到国际PAH突变数据库（www.pahdb.mcgill.ca）。突变基因的总频率为30.61%（90 /294）。突变涉及了错义、缺失、移码和剪接位点4种突变类型。结果明确了PAH基因外显子7的突变种类和分布等特征,表明外显子7是中国人PAH基因突变的热点区域。 Abstract: To study mutation in exon 7 of the gene for the phenylalanine hydroxylase（PAH）, the mutations in exon 7 and flanking sequence of PAH gene were detected by means of SSCP analysis and DNA sequencing, in 147 unrelated Chinese children with phynelketonuria and their parents. Thirteen different mutations, including 11 missense, 1 deletion and 1 splice mutation, were revealed in 90/294 mutant alleles (30.61%). The prevalent mutations were R243Q (22.8%) and Ivs7nt2t->a (2.38%). Seven novel mutations were identified: G239D, R241fsdelG, G247S, E280G, L255S, R261Q, P281L. These new mutations have not been described in Chinese PKU population and the first 4 mutants have not been reported and thus been submitted to www.pahdb,mcgill.ca. The missense was the most common type. The deletion and frameshift mutations were detected for the first time in Chinese PKU population. This study showed the mutation characteristics and their distribution in exon 7 of PAH gene and proved that the exon 7 was the hot region of PAH gene mutation in Chinese PKU population .     

Phenylketonuria mutations in Germany

We report the spectrum of mutations and associated modified haplotypes in patients with phenylketonuria living in Germany. A total of 546 independent alleles was investigated, including 411 of German and 65 of Turkish descent. Mutations were identified for 535 PKU alleles (98%) and there were 91 different mutations. The most common mutation was R408W on 22% of alleles. Two mutations, IVS12+1G→A and IVS10–11G→A accounted for just under 10% of alleles, whereas the remaining mutations were found at relative frequencies of 6% or less; 43 mutations were observed once only. IVS10–11G→A was the most common mutation (38% of alleles) in the subgroup of patients of Turkish descent. Modified haplotypes were determined from the analysis of four silent mutations, three diallelic restriction fragment length polymorphisms, a variable number of tandem repeats minisatellite and a short tandem repeat microsatellite in the phenylalanine hydroxylase gene, showing that a considerable proportion of mutations must have recurred in independent founders; other mutations may have changed chromosomal haplotype backgrounds by gene conversion. The spectrum of PKU mutations in Germany reflects the history of a heterogenous Central European population living at the crossroads of migration throughout the centuries. Received: 11 January 1999 / Accepted: 11 March 1999     

PKU in Slovakia: mutation screening and haplotype analysis

The restriction fragment length polymorphism haplotypes and seven common mutations in the phenylalanine hydroxylase gene were analysed in 49 unrelated Slovak phenylketonuria (PKU) families of Caucasian origin. The predominant mutation in this population sample is R408W, with a frequency of 45.9%. In addition, four other mutations have been identified at relatively high frequencies: IVS12nt1, 10.2%; R158Q, 7.1%; R261Q, 7.1%; R252W, 2.0%. The mutation-haplotype associations correspond to those described in other European populations. The high proportion of mutations (72.4%) amenable to simple rapid detection based on the polymerase chain reaction provides a good basis for direct DNA-diagnosis of PKU in the Slovak population.     

Mutation spectrum of the PAH gene in the PKU patients from Khorasan Razavi province of Iran

Background

Characterization of the molecular basis of phenylketonuria (PKU) in North-east of Iran has been accomplished through the analysis of 62 unrelated chromosomes from 31 Iranian PKU patients.

Methods

Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by direct DNA sequencing exons 6, 7, 10 and 11.

Results

A mutation detection rate of 74% was achieved. Eleven different mutations were found, with the most frequent mutation, IVS10-11G > A, accounting for 19% of Khorasan-Razavi PKU alleles. Ten mutations (R176X, E280K, IVS11 + 1G > C, S231P, Q383X, R243X, I224T, E390G, R252W and P281L) represent the rest PKU chromosomes. One novel mutation, Q383X in the homozygote form was identified which is located in the catalytic domain (residues143–410).

Conclusion

With this high detection rate of mutations in North-east of Iran, new strategy for carrier testing could be DNA sequencing of these four exons. The other exons and boundaries will be studied only when either one or no mutations are detected in the initial screen.     

Analysis of phenylalanine hydroxylase gene mutations in phenylketonuria patients from Kemerovo oblast and the Sakha Republic

This paper presents the results of a molecular genetic study on the phenylalanine hydroxylase (PAH) gene among phenylketonuria (PKU) patients and their family members residing in Kemerovo oblast and the Sakha Republic. To reveal the PAH gene mutations, the researchers applied exon amplification and a direct determination of their nucleotide sequences. The study has revealed both well-known mutations (R158Q, R252W, R261Q, P281L, IVS10 ? 11G > A, R408W, and IVS12 + 1G > A) and some rarely encountered ones (IVS2 + 5G > A, R155H, Y168H, W187R, E221-D222 > Efs, A342T, Y386C, and IVS11 + 1G > C). Some of the studied populations with a mixed ethnic ancestry have been shown to demonstrate a wider spectrum of their PKU-associated alleles.     

Polymorphic DNA haplotypes at the phenylalanine hydroxylase (PAH) locus in European families with phenylketonuria (PKU)

DNA haplotype data from the phenylalanine hydroxylase (PAH) locus are available from a number of European populations as a result of RFLP testing for genetic counseling in families with phenylketonuria (PKU). We have analyzed data from Hungary and Czechoslovakia together with published data from five additional countries--Denmark, Switzerland, Scotland, Germany, and France--representing a broad geographic and ethnographic range. The data include 686 complete chromosomal haplotypes for eight RFLP sites assayed in 202 unrelated Caucasian families with PKU. Forty-six distinct RFLP haplotypes have been observed to date, 10 unique to PKU-bearing chromosomes, 12 unique to non-PKU chromosomes, and the remainder found in association with both types. Despite the large number of haplotypes observed (still much less than the theoretical maximum of 384), five haplotypes alone account for more than 76% of normal European chromosomes and four haplotypes alone account for more than 80% of PKU-bearing chromosomes. We evaluated the distribution of haplotypes and alleles within these populations and calculated pairwise disequilibrium values between RFLP sites and between these sites and a hypothetical PKU "locus." These are statistically significant differences between European populations in the frequencies of non-PKU chromosomal haplotypes (P = .025) and PKU chromosomal haplotypes (P much less than .001). Haplotype frequencies of the PKU and non-PKU chromosomes also differ significantly (P much less than .001. Disequilibrium values are consistent with the PAH physical map and support the molecular evidence for multiple, independent PKU mutations in Caucasians. However, the data do not support a single geographic origin for these mutations.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Development of a mutation hotspot detection kit for the phenylalanine hydroxylase gene by ARMS-PCR combined with fluorescent probe technology

To develop a screening kit for detecting mutation hotspots of the phenylalanine hydroxylase (PAH) gene. Thirteen exons of the PAH gene were sequenced in 84 cases with phenylketonuria (PKU) diagnosed during neonatal genetic and metabolic disease screening in Shaanxi province, and their mutations were analyzed. We designed and developed a screening kit to detect nine mutation sites covering more than 50% of the PAH mutations found in Shaanxi province (c.728G>A, c.1197A>T, c.331C>T, c.1068C>A, c.611A>G, c.1238G>C, c.721C>T, c.442-1G>A, and c.158G>A) by using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) combined with fluorescent probe technology. Peripheral blood and dried blood samples from PKU families were used for clinical verification of the newly developed kit. PAH gene mutations were detected in 84 children diagnosed with PKU. A total of 159 mutant alleles were identified, consisting of 100 missense mutations, 28 shear mutations, 24 nonsense mutations, and 7 deletion mutations. Exon 7 had the highest mutation frequency (32.08%). Among them, the mutation frequency of p.R243Q was the highest, accounting for 20.13% of all mutations, followed by p.R111X, IVS4-1G>A, EX6-96A>G, and p.R413P; these five loci accounted for 47.17% (75/159) of all mutations. In addition, we identified three previously unreported PAH gene mutations (p.C334X, p.G46D, and p.G256D). Fifteen mutation sites were identified in the 47 PAH carriers identified by next-generation sequencing (NGS), which were verified by the newly developed kit, with an agreement rate of 100%. This newly developed kit based on ARMS-PCR combined with fluorescent probe technology can be used to detect common PAH gene mutations.