Mutation spectrum in Taiwanese patients with phenylalanine hydroxylase deficiency and a founder effect for the R241C mutation

The spectrum of phenylalanine hydroxylase (PAH) gene mutations was determined in 25 families of hyperphenylalaninemia identified by a neonatal screening program in Taiwan. The coding sequence and exon-flanking intron sequences of PAH gene were amplified and sequenced. Mutations were identified in forty-five of the 50 chromosomes. R241C was the most common mutation (36% of the chromosomes), followed by R408Q (14% of the chromosomes). The remaining mutations were rare and seven mutations have not been reported before: p.F233L (c.697T>C), p.R252Q (c.756G>A), p.E286K (c.856G>A), p.G312V (c.935G>T), p.P314T (c.940C>A), p.I95del (c.284_286delTCA), and p.T81fsX6 (c.241_256del). Both p.R241C and p.R408Q are classified as mild phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) mutation, which may explain the fact that classical PKU is very rare in Taiwan (n=4, or one in 413,035). This strong founder effect for the p.R241C mutation has been described neither in the Caucasian populations, nor in other reports from Chinese. Since most of the populations in Taiwan are derived from Southeastern China, the spectrum of PAH gene mutations in Southeastern China should be different from other Chinese populations. This report not only disclose a specific spectrum of PAH gene mutation in Taiwan, but may also give clues to the movement of populations in Mainland China.     

McArdle disease: the mutation spectrum of PYGM in a large Italian cohort

Deficiency of the muscle isozyme of glycogen phosphorylase is causative of McArdle disease or Glycogen storage disease type V (GSD-V), the most common autosomal recessive disorder of glycogen metabolism. The typical clinical presentation is characterized by exercise intolerance with cramps, and recurrent myoglobinuria. To date, 46 mutations in the PYGM gene have been detected in GSD-V patients. We report the mutational spectrum in 68 Italian patients. We identified 30 different mutations in the PYGM gene, including 19 mutations that have not been reported previously. The novel mutations include: eight missense mutations (c.475G>A, p.G159R; c.689C>G, p.P230R; c.1094C>T, p.A365E; c.1151C>A, p.A384D; c.1182C>T, p.R428C; c.1471C>T, p.R491C; c.2444A>C, p.D815A; c.2477G>C, p.W826S), two nonsense mutations (c.1475G>A, p.W492X; c.1627A>T, p.K543X), five splice site mutations (c.855 +1G>C; c.1092 +1G>A; c. 1093-1G>T; c.1239 +1G>A; c.2380 +1G>A), and four deletions (c.715_717delGTC, p.V239del; c.304delA, p.N102DfsX4; c.1970_2177del, p.V657_G726; c.2113_2114delGG, p.G705RfsX16). Whereas we confirmed lack of direct correlation between the clinical phenotype and the genotype, we also found that the so-called 'common mutation' (p.R50X) accounted for about 43% of alleles in our cohort and that no population-related mutations are clearly identified in Italian patients.     

The activity of wild-type and mutant phenylalanine hydroxylase and its regulation by phenylalanine and tetrahydrobiopterin at physiological and pathological concentrations: an isothermal titration calorimetry study

The activity of phenylalanine hydroxylase (PAH) is regulated by the levels of both the substrate (L-Phe) and the natural cofactor (6R)-tetrahydrobiopterin (BH4). It has recently been observed that many PAH mutants associated with BH4-responsive phenylketonuria display abnormal kinetic and regulatory properties as shown by standard kinetic analyses. In this work, we have developed a high-sensitive and high-throughput activity assay based on isothermal titration calorimetry (ITC) in order to study the kinetic properties of wild-type PAH (wt-PAH) and the BH4-responsive c.204A>T (p.R68S) mutant at physiological and superphysiological concentrations of L-Phe and BH4. Compared to wt-PAH, the p.R68S mutant showed reduced apparent and equilibrium binding affinity for the natural cofactor and increased affinity and non-cooperative response for L-Phe, together with a strong substrate inhibition that is alleviated at high BH4 concentrations. For both wt-PAH and mutant, the apparent affinity for BH4 decreases at increasing L-Phe concentrations, and the affinity for the substrate also depends on the cofactor concentration. Our results indicate that the activity landscape for wt and mutant enzymes is more complex than expected from standard kinetic analyses and highlight the applicability of this ITC-based assay to characterize the activity and regulation of PAH at a wide range of substrate and cofactor concentrations. Moreover, the results aid to understand the activity dynamics of wild-type and mutant PAH under physiological and pathological conditions, as well as BH4-responsiveness in certain PKU mutations.     

Molecular genetics of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

Mutations in the phenylalanine hydroxylase (PAH) gene result in phenylketonuria (PKU). Tetrahydrobiopterin (BH(4))-responsive hyperphenylalaninemia has been recently described as a variant of PAH deficiency caused by specific mutations in the PAH gene. It has been suggested that BH(4)-responsiveness may be predicted from the corresponding genotypes. Data from BH(4) loading tests indicated an incidence of BH(4)-responsiveness of >40% in the general PKU population and >80% in mild PKU patients. The current project entailed genotype analysis of 315 BH(4)-responsive patients tabulated in the BIOPKUdb database and comparison with the data from the PAHdb locus-specific knowledgebase, as well as with previously published PAH mutations for several European countries, Northern China, and South Korea. We identified 57 mutations, presenting with a substantial residual PAH activity (average approximately 47%), presumed to be associated with BH(4)-responsiveness. More than 89% of patients are found to be compound heterozygotes. The three most common mutations found in >5% of BH(4)-responsive patients are p.A403 V, p.R261Q, and p.Y414C. Using the Hardy-Weinberg formula the predicted average frequency of BH(4)-responsiveness in European populations was calculated to be 55% (range 17-79%, lowest in Baltic countries and Poland and highest in Spain), 57% in Northern China, and 55% for South Korea. The genotype-predicted prevalence of BH(4)-responsiveness was higher than prevalence data obtained from BH(4) loading tests. Inconsistent results were observed for mutations p.L48S, p.I65 T, p.R158Q, p.R261Q, and p.Y414C. Our data suggest that BH(4)-responsiveness may be more common than assumed and to some extent may be predicted or excluded from the patient's genotype.     

Functional assays testing pathogenicity of 14 cystathionine-beta synthase mutations

In this study, 14 CBS alleles from homocystinuric patients were expressed heterologously in E. coli and their enzyme activities were assayed in vitro. Additionally, mutant CBS proteins were visualized by Western blot from denaturing and non-denaturing polyacrylamide gels. The 14 mutations characterized were: p.R125W (c.373C>T), p.G148R (c.442G>A), p.M173V (c.517A>G), p.T191M (c.572C>T), p.A226T (c.676G>A), p.C275Y (c.824G>A), p.R336C (c.1006C>T), p.R336H (c.1007G>A), p.L338P (c.1013T>C), p.S349N (c.1046G>A), p.R379Q (c.1136G>A), p.L456P (c.1367T>C), p.G522fsX540 (c.1566delG), and p.R548Q (c.1643G>A). Eleven of the mutant alleles exhibited an activity lower than 4% of the wild-type protein. In contrast, mutations p.A226T and p.M173V presented 20% and 40% of the wild-type activity, respectively, whereas the activity of p.R548Q was up to 60% of the wild-type. This suggests that it is a new rare variant rather than a pathogenic mutation. Most of the mutated proteins exhibited a decreased signal in Western blot analyses. The non-denaturing PAGE revealed that the wild-type protein retained the capacity to form a multimeric quaternary structure, whereas in the mutations p.M173V, p.A226T, and p.G548Q, this structure grade was dramatically reduced and was completely absent in the rest of the mutations.     

Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable: molecular pathology of mutations in PAH exon 11

In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation molecular pathology is important. Mutations that disturb the splicing of exons (e.g. interplay between splice site strength and regulatory sequences like exon splicing enhancers (ESEs)/exon splicing silencers (ESSs)) may cause different severity of PKU. In this study, we identified PAH exon 11 as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T>C mutation resides in a putative splicing enhancer motif and binding by splicing factors SF2/ASF, SRp20 and SRp40 is disturbed. Additional mutations in potential splicing factor binding sites contributed to elucidate the pathogenesis of mutations in PAH exon 11. We suggest that PAH exon 11 is vulnerable due to a weak 3' splice site and that this makes exon 11 inclusion dependent on an ESE spanning position c.1144. Importantly, this implies that other mutations in exon 11 may affect splicing, since splicing is often determined by a fine balance between several positive and negative splicing regulatory elements distributed throughout the exon. Finally, we identified a pseudoexon in intron 11, which would have pathogenic consequences if activated by mutations or improved splicing conditions. Exonic mutations that disrupt splicing are unlikely to facilitate response to BH(4) and may lead to inconsistent genotype-phenotype correlations. Therefore, recognizing such mutations enhances our ability to predict the BH(4)-response.     

中国北方地区苯丙氨酸羟化酶基因的突变构成

目的了解中国人苯丙氨酸羟化酶（phenylalanine hydroxylase,PAH）基因的突变构成。方法应用PCR单链构象多态结合序列分析检测230例苯丙酮尿症患儿PAH基因全部外显子及其两侧内含子。结果（1）在460个PAH等位基因中检测出75种不同的突变基因,总检测率达94.6%（435/460）,其中3种突变基因（S251-R252〉SfsX89、Y387D和A389G）尚未见到报道。常见的突变基因为：R243Q（21.7%）、EX6-96A〉G（10.2%）、R111X（8.3%）、R413P（6.5%）和Y356X（6.1%）。较常见的突变基因为：V399V（4.1%）、IVS4-1G〉A（3.5%）、IVS7＋2T〉A（2.2%）和R241C（2.2%）。大部分突变集中在第3、5、6、7、11和12外显子及其两侧内含子区域。（2）检测出10种多态性位点,突变率高的4个位点IVS3-22C〉T（56.7%）、IVS10＋97G〉A（75.9%）、Q232Q（89.0%）和V245V（81.9%）,提示PAH基因cDNA序列存在人种差异。结论中国人PAH基因的突变构成与欧洲人群完全不同,与亚洲其它人群有频率的差异。     

Identification of the bruton tyrosine kinase (BTK) gene mutations in 20 Australian families with X-linked agammaglobulinemia (XLA)

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the Bruton tyrosine kinase (BTK) gene. Twenty Australian patients with an XLA phenotype, from 15 unrelated families, were found to have 14 mutations. Five of the mutations were previously described c.83G>A (p.R28H), c.862C>T (p.R288W), c.904G>A (p.R302G), c.1535T>C (p.L512P), c.700C>T (p.Q234X), while nine novel mutations were identified: four missense c.82C>A (p.R28S), c.494G>A (p.C165Y), c.464G>A (p.C155Y), c.1750G>A (p.G584E), one deletion c.142_144delAGAAGA (p.R48_G50del), and four splice site mutations c.241-2A>G, c.839+4A>G, c.1350-2A>G, c.1566+1G>A. Carrier analysis was performed in 10 mothers and 11 female relatives. The results of this study further support the notion that molecular genetic testing represents an important tool for definitive and early diagnosis of XLA and may allow accurate carrier status and prenatal diagnosis.     

Mutational spectrum in German patients with phenylalanine hydroxylase deficiency

We report on the spectrum and frequency of mutations in the phenylalanine hydroxylase (PAH) gene in 226 German families with PAH deficiency, most of them from Southern Germany. A total of 88 mutations were identified in 428 out of 438 mutant PAH alleles including one novel stop mutation L293X (c.878T>A). In three families, two phenylketonuria (PKU) mutations were found in cis, and in one family a de novo mutation was observed. A comparison of the results from Southern Germany with those of other parts of Western Germany showed no obvious local mutation clustering. In addition we studied the genotypic spectrum of 39 Turkish families with PAH deficiency. Twenty-three mutations were identified in 73 out of 75 Turkish chromosomes including two novel mutations: E280A (c.839A>G) in Exon 7 and IVS10-7C>A (c.1066-7C>A) in Intron 10. A new polymorphism IVS4+47C/T (c.441+47C>T) was found in mutant and normal PAH alleles. Screening of 170 German and 150 Turkish individuals without family history of PAH deficiency revealed 10 and 12 heterozygotes, respectively, a higher frequency of carriers than expected. A novel mutations of uncertain functional relevance, R169H (c.506G>A) in Exon 5 was found in two Turkish heterozygotes. Most of the Turkish heterozygotes carried mild mutations, indicating that mild forms of PAH deficiency may be more common in that population than previously recognised.     

High prevalence of mutations in LCAT in patients with low HDL cholesterol levels in The Netherlands: identification and characterization of eight novel mutations

Lecithin:cholesterol acyltransferase (LCAT) is crucial to the maturation of high-density lipoprotein (HDL). Homozygosity for LCAT mutations underlies rare disorders characterized by HDL-cholesterol (HDL-c) deficiency while heterozygotes have half normal HDL-c levels. We studied the prevalence of LCAT mutations in referred patients with low HDL-c to better understand the molecular basis of low HDL-c in our patients. LCAT was sequenced in 98 patients referred for HDL-c <5th percentile and in four patients referred for low HDL-c and corneal opacities. LCAT mutations were highly prevalent: in 28 of the 98 participants (29%), heterozygosity for nonsynonymous mutations was identified while 18 patients carried the same mutation (p.T147I). The four patients with corneal opacity were compound heterozygotes. All previously identified mutations are documented to cause loss of catalytic activity. Nine novel mutations-c.402G>T (p.E134D), c.403T>A (p.Y135N), c.964C>T (p.R322C), c.296G>C (p.W99S), c.736G>T (p.V246F), c.802C>T (p.R268C), c.945G>A (p.W315X), c.1012C>T (p.L338F), and c.1039C>T (p.R347C)--were shown to be functional through in vitro characterization. The effect of several mutations on the core protein structure was studied by a three-dimensional (3D) model. Unlike previous reports, functional mutations in LCAT were found in 29% of patients with low HDL-c, thus constituting a common cause of low HDL-c in referred patients in The Netherlands.     

Identification of 25 new mutations in 40 unrelated Spanish Niemann-Pick type C patients: genotype-phenotype correlations

To better characterize Niemann-Pick type C (NPC) in Spain and improve genetic counselling, molecular analyses were carried out in 40 unrelated Spanish patients. The search identified 70/80 alleles (88%) involving 38 different NPC1 mutations, 26 of which are described for the first time. No patient with NPC2 mutations was identified. The novel NPC1 mutations include 14 amino acid substitutions [R372W (c.1114C>T), P434L (c.1301C>T), C479Y (c.1436G>A), K576R (c.1727G>A), V727F (c.2179G>T), M754K (c.2261T>A), S865L (c.2594C>T), A926T (c.2776G>A), D948H (c.2842G>C), V959E (c.2876T>A), T1036K (c.3107C>A), T1066N (c.3197C>A), N1156I (c.3467A>T) and F1224L (c.3672C>G)], four stop codon [W260X (c.780G>A), S425X (c.1274C>A), C645X (c.1935T>A) and R1059X (c.3175C>T)], two donor splice-site mutations [IVS7+1G>A (g.31432G>A) and IVS21+2insG (g.51871insG)], one in-frame mutation [N961_F966delinsS (c.2882del16bpins1bp)] and five frameshift mutations [V299fsX8 (c.895insT), A558fsX11 (c.1673insG), C778fsX10 (c.2334insT), G993fsX3 (c.2973_78delG) and F1221fsX20 (c.3662delT)]. We also identified three novel changes [V562V (c.1686G>A), A580A (c.1740C>G) and A1187A (c.3561G>T)] in three independent NPC patients and five polymorphisms that have been described previously. The combination of these polymorphisms gave rise to the establishment of different haplotypes. Linkage disequilibrium was detected between mutations C177Y and G993fsX3 and specific haplotypes, suggesting a unique origin for these mutations. In contrast, I1061T mutation showed at least two different origins. The most prevalent mutations in Spanish patients were I1061T, Q775P, C177Y and P1007A (10, 7, 7 and 5% of alleles, respectively). Our data in homozygous patients indicate that the Q775P mutation correlates with a severe infantile neurological form and the C177Y mutation with a late infantile clinical phenotype.     

Mutational analysis of mucopolysaccharidosis type VI patients undergoing a phase II trial of enzyme replacement therapy

Mucopolysaccharidosis type VI (MPS VI; Maroteaux-Lamy syndrome) is a lysosomal storage disorder caused by mutations in the N-acetylgalactosamine-4-sulfatase (ARSB) gene. These mutations result in a deficiency of ARSB activity. Ten MPS VI patients were involved in a phase II clinical study of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each individual exon of the ARSB gene was amplified by PCR and subsequently sequenced. Thirteen substitutions (c.215T>G [p.L72R] c.284G>A [p.R95Q], c.305G>A [p.R102H], c.323G>T [p.G108V], c.389C>T [p.P130L], c.511G>A [p.G171S], c.904G>A [p.G302R], c.944G>A [p.R315Q], c.1057T>C [p.W353R], c.1151G>A [p.S384N], c.1178A>C [p.H393P], c.1289A>G [p.H430R] and c.1336G>C [p.G446R]), one deletion (c.238delG), and two intronic mutations (c.1213+5G>A and c.1214-2A>G) were identified. Nine of the 16 mutations identified were novel (R102H, G108V, P130L, G171S, W353R, H430R, G446R, c.1213+5G>A and c.1214-2A>G). The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified in some of the patients, along with the silent mutations c.972A>G and c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient and, together with genotype information, used to predict the expected clinical severity of each patient.     

Identification of novel MLC1 mutations in Chinese patients with megalencephalic leukoencephalopathy with subcortical cysts (MLC)

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal, recessively inherited disease caused by mutations in the MLC1 gene. Most of the previously published studies have been carried out in ethnic populations other than the Chinese. In this study, the analysis of clinical features and MLC1 mutation screening were performed in 13 Chinese patients for the first time. A total of 10 MLC1 mutations were identified in these patients, including five novel missense mutations (c.65G>A, p.R22Q; c.95C>T, p.A32V; c.218G>A, p.G73E; c.823G>A, p.A275T; c.832T>C, p.Y278H), one novel splicing mutation (c.772-1G>C in IVS9-1), one novel small deletion (c.907_930del, p.V303_L310del), one known nonsense mutation (c.593delCTCA, p.Y198X) and two known missense mutations (c.206C>T, p.S69L; c.353C>T, p.T118M). Mutation c.772-1G>C in IVS9-1, accounting for 27.3% (3/11) of the total number of genetically confirmed patients found in this study, is thus a putative hot-spot mutation in the present study group. The existence of a unique MLC1 mutation spectrum in Chinese MLC patients was shown. A systemic study to assess the mutation spectra in different populations should be undertaken.     

Mutation spectrum of phenylketonuria in Iranian population

Identification of molecular basis of phenylketonuria (PKU) in Iran has been accomplished through the analysis of 248 unrelated chromosomes from 124 Iranian classic PKU subjects. Phenylalanine hydroxylase (PAH) gene mutations were analyzed through a combined approach in which p.S67P, p.R252W, p.R261Q, p.R261X, p.L333F, IVS10-11G>A, IVS11+1G>C, p.L364del, p.R408Q and p.R408W mutations were first screened by PCR of PAH gene exons 3, 7, 10, 11 and 12, followed by digestion with the appropriate digestion enzymes. Subsequently SSCP analysis for exons 2, 6, 7 and 11 of the PAH gene and finally, sequencing of 13 PAH gene exons have been used to study uncharacterized PKU chromosomes. 26 different mutations were found. The predominant mutation in this population sample was IVS10-11G>A, with a frequency of 24.6%. Nine mutations (IVS10-11G>A, p.R261Q, p.P281L, IVS11+1G>C, p.K363>NFS, p.R243X, IVS2+5G>C, p.R261X and p.R252W) represent almost 84% of all PKU chromosomes studied. IVS10-11G>A mutation is the major PKU-causing mutation throughout the Mediterranean region. The finding of the high prevalence of this mutation in Iranian population is consistent with the historical and geographical links between Iranian and Mediterranean populations.     

Clinical and nutritional evaluation of phenylketonuric patients on tetrahydrobiopterin monotherapy

The clinical, nutritional, and neuropsychological data of 11 mild/moderate PKU patients after one year of treatment with BH4 are evaluated. BH4 monotherapy was introduced at 5 mg/kg/day in 14 PKU patients. In 11/14 patients, Phe tolerance increased significantly from 356+/-172 to 1546+/-192 mg/day (p=0.004), and special PKU formula was gradually reduced until complete removal. In them, mean plasma Phe concentrations remained below 360 micromol/L at 5 mg BH4/kg/day (7 mg/kg/day in one patient). BH4 therapy was stopped in three patients (V388M/P362T and R243Q/IVS10-11G>A genotypes) because it was not possible to improve Phe tolerance and to remove formula intake. Serum micronutrients were not significantly different at the start of treatment and at one year follow-up, except for selenium, which increased significantly after one year of therapy (p=0.017). Anthropometric, and nutritional measurements were within the age- and sex-specific percentiles for a healthy population after one year therapy. Neuropsychological follow-up indicated that intelligence scores persisted within normal limits. In terms of patients' genotype, we confirmed that the P275S mutation combined with R408W was associated with long-term BH4 responsiveness, while the combination of P362T/V388M, and R243Q/IVS10-11G>A resulted in poor metabolic control in long-term BH4 therapy. In summary, our data confirm that BH4 is a safe, and effective therapy in a selected group of mild, and moderate PKU patients who respond to the BH4 loading test. Low doses of BH4 in monotherapy permit withdrawal of the special formula and guarantee a good clinical and nutritional outcome with no adverse side effects in PKU patients.     

Mutation analysis of seven patients with Waardenburg syndrome北大核心CSCD

Objective: To perform genetic analysis for 7 patients with Waardenburg syndrome. Methods: Potential mutation of MITF, PAX3, SOX10 and SNAI2 genes was screened by polymerase chain reaction and direct sequencing. Functions of non-synonymous polymorphisms were predicted with Polyphen2 software. Results: Seven mutations, including c. 649-651delAGA (p. R217del), c. 72delG (p. G24fs), c. 185T>C (p. M62T), c. 118C>T (p. Q40X), c. 422T>C (p. L141P), c. 640C>T (p. R214X) and c. 28G>T (p. G43V), were detected in the patients. Among these, four mutations of the PAX3 gene (c. 72delG, c. 185T>C, c. 118C>T and c. 128G>T) and one SOX10 gene mutation (c. 422T>C) were not reported previously. Three non-synonymous SNPs (c. 185T>C, c. 128G>T and c. 422T>C) were predicted as harmful. Conclusion: Genetic mutations have been detected in all patients with Waardenburg syndrome. © 2016, West China University of Medical Sciences. All rights reserved.     

Five novel mutations and two large deletions in a population analysis of the phenylalanine hydroxylase gene

Mutational spectrum of the phenylalanine hydroxylase (PAH) deficiency was investigated in 107 families (90% of the Slovene PKU population). The entire coding region of the PAH gene was analyzed with dHPLC to select the samples where subsequently the automated sequencing analysis was performed. MLPA analysis was performed to identify large deletions, which were later confirmed with long-range PCR. Correlations with patients' phenotypes and genotype-based predictions of BH(4)-responsiveness were assessed. Altogether, disease-causing mutations were identified on 209 alleles (detection rate 97.7%). A spectrum of 36 different disease-causing mutations was identified: 20 missense mutations (80% of the alleles), eight splicing mutations (13% of the alleles), one nonsense mutation (0.5% of the alleles), four small deletions with frame shift (6% of the alleles), one small insertion with frame shift (0.5% of the alleles), and two large deletions (2% of the alleles). The most frequent mutation was p.R408W in exon 12, representing 29% of the alleles, which is in concordance with other neighboring and/or Slavic PKU populations. Other common mutations were: p.R158Q, p.A403V, p.P281L and p.E390G, accounting for 9%, 7%, 7% and 7% of the alleles respectively. Five novel mutations were detected: c.43_44insAG, c.56_59+1delACAGG, p.V45A, p.L62P and p.R157S. Large deletion of exon 5 (EX5del955) was found in three patients and a deletion of exon 3 (EX3del4765) in one patient. A spectrum of 64 different genotypes was found, seven of them accounting for over than a third of all families. Among thirteen families with homozygous mutation (13% of the PKU population), 10 had p.R408W, two had p.R158Q and one had p.E390G. Among 107 families, 58 were classified as classic PKU (54.2%), 28 as mild PKU (25.9%) and 21 as MHP (19.6%). Twenty-six different genotypes (40.6%) were predicted to be BH(4)-responsive, represented by 38 different families (35.5%).     

Molecular genetics of tetrahydrobiopterin (BH4) deficiency in the Maltese population

Deficient activity of the Dihydropteridine Reductase enzyme (DHPR; EC 1.5.1.34; OMIM 261630) is due to mutations in the Quinoid Dihydropteridine Reductase gene on 4p15.3 (QDPR; RefSeq NM_000320). It results in defective recycling of tetrahydrobiopterin (BH(4)) and homozygotes have a rare form of atypical Hyperphenylalaninaemia and Phenylketonuria (aPKU). The heterozygote frequency in the Maltese population is high at 3.3%. The more recently described and rarer type of BH(4) deficiency due to Sepiapterin Reductase enzyme deficiency (SR; EC 1.1.1.153; OMIM 182125), which presents as an atypical form of Dopa Responsive Dystonia (DRD) [L. Bonafe, B. Thony, J.M. Penzien, B. Czarnecki, N. Blau, Mutations in the sepiapterin reductase gene cause a novel tetrahydrobiopterin-dependent monoamine-neurotransmitter deficiency without hyperphenylalaninemia, Am. J. Hum. Genet. 69 (2001) 269-277; B.R.G. Neville, R. Parascandalo, S. Attard Montalto, R. Farrugia, A.E. Felice, A congenital dopa responsive motor disorder: a Maltese variant due to sepiapterin reductase deficiency, Brain 128 (Pt10) (2005) 2291-2296.] has also been identified at high frequency (4.6%) in this population. Two mutations, the c.68G>A in QDPR (p.G23D), and the new SPR, IVS2-2A>G mutation at the splice site consensus sequence in intron 2 of the Sepiapterin Reductase gene (SPR; RefSeq NM_003124) on 2p14-p12, were found to be the sole causative mutations in all the patients with DHPR deficiency and SR deficiency studied. All parents were heterozygotes for the corresponding mutation and showed no clinical symptoms. Three polymorphisms, c.96C>T (p.A32A), c. 345G>A (p.S115S) and c. 396G>A (p.L132L), have also been identified in the QDPR gene, defining four wild-type frameworks, useful in molecular epidemiology studies. The c. 68G>A mutation in QDPR was found only on framework I, suggesting a founder effect. In contrast no additional sequence diversity was found in the SPR gene whether in wild-type or mutant alleles which is also consistent with a founder effect.     

Mutational analysis of mucopolysaccharidosis type VI patients undergoing a trial of enzyme replacement therapy

Mucopolysaccharidosis type VI (MPS VI), or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (ARSB). Seven MPS VI patients were chosen for the initial clinical trial of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each individual exon of the ARSB gene was amplified by PCR and subsequently sequenced. Nine substitutions (c.289C>T [p.Q97X], c.629A>G [p.Y210C], c.707T>C [p.L236P], c.936G>T [p.W312C], c.944G>A [p.R315Q], c.962T>C [p.L321P], c.979C>T [p.R327X], c.1151G>A [p.S384N], and c.1450A>G [p.R484G]), two deletions (c.356_358delTAC [p.Y86del] and c.427delG), and one intronic mutation (c.1336+2T>G) were identified. A total of 7 out of the 12 mutations identified were novel (p.Y86del, p.Q97X, p.W312C, p.R327X, c.427delG, p.R484G, and c.1336+2T>G). Two of these novel mutations (p.Y86del and p.W312C) were expressed in Chinese hamster ovary cells and analyzed for residual ARSB activity and mutant ARSB protein. The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified among the patients, along with the silent mutation c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient, and, together with genotype information, were used to predict the expected clinical severity of each MPS VI patient.     

Mucopolysaccharidosis I mutations in Chinese patients: identification of 27 novel mutations and 6 cases involving prenatal diagnosis

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease that results from the deficiency of α-l-iduronidase and is transmitted in an autosomally recessive manner. This report describes the first systematic screening for mutations in Chinese MPS I patients from mainland China, wherein we have summarized the phenotype/genotype correlation of the individuals in Chinese MPS I patients. Mutational analyses were performed in 57 unrelated Chinese MPS I patients. Overall, 105 mutant alleles were identified from a set of 41 different mutations. Notably, of these 41 mutations, 27 were novel mutations that consisted of 8 splicing mutations (c.1-2C>G, c.296+4G>A, c.300-1G>C, c.792+1G>C, c.973-4G>A, c.1189+5G>T, c.1402+1G>T and c.1402+2T>G), 1 nonsense mutation (p.W41X), 1 insertion (c.668-670ins GCG), 5 duplications (c.531dupT, c.657dupG, c.883dupC, c.1147dupG and c.1225dupG), 3 deletions (c.349delT, c.1593delG and c.1244-1271del27),1 nucleotide substitution c.2T>C and 8 missense mutations (p.H33P,p.F52L, p.G168V,p.T179R,p. E182D, p.L237R, p.L238R and p.L421P). The missense mutations p.A79V and p.L346R, which accounted for 16.7% (19/114) and 12.3% (14/114) respectively, were the common mutations in Chinese patients but were rare in the mutational profiles reported for other populations. These results indicate that Chinese MPS I patients may have a different mutational spectrum compared to those of other populations. Moreover, for the first time in China, molecular genetic methods were used for prenatal diagnosis of six cases in five families.