Abstract
Hyperphenylalaninemia (HPA) is a condition caused by tetrahydrobiopterin (BH4) and phenylalanine hydroxylase (PAH) deficiencies. It is essential that differential diagnosis be conducted to distinguish these two causes of HPA, because BH4 deficiency is a more severe disease involving progressive neurologic deterioration. Based on the biological findings, HPA is defined as a plasma phenylalanine level of >2.0 mg/dl (>120 μmol/l). The National Biochemistry Reference Laboratory at the Pasteur Institute of Iran initiated BH4 deficiency screening tests for the first time during the implementation of a nationwide phenylketonuria (PKU) screening program. Measurement of blood phenylalanine and urinary neopterin and biopterin was conducted by high-performance liquid chromatography in 617 patients with HPA. Dihydropteridine reductase (DHPR) activity was measured in all patients by kinetic spectrophotometry. Differential diagnosis was conducted for PKU, transient HPA, and BH4 deficiencies.
Our results indicated that out of 76 cases involving BH4 deficiencies, 37 had 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, 35 had DHPR deficiency, 1 case had pterin-4a-carbinolamine dehydratase (PCD) deficiency, and 3 cases had GTP cyclohydrolase I (GTPCH) deficiency. In this study, 1 novel deletion mutation and 18 novel missense mutations were reported in addition to mutations that had previously been identified and registered in BIOMDB. At present, the screening program for PKU in Iran includes tests that detect different forms of BH4 deficiency presenting with HPA. Newborns that are BH4-deficient benefit from the availability of the tests because they can receive necessary care before being clinically affected.
Keywords: BH4 deficiencies, Hyperphenylalaninemia, Phenylketonuria
Introduction
The fully functional phenylalanine hydroxylase (PAH) system for phenylalanine (Phe) metabolism consists of the PAH (EC 1.14.16.1) enzyme, the cofactor tetrahydrobiopterin (BH4), and two regenerating enzymes: pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) (Blau et al. 2010). PAH deficiency and BH4 deficiency result in hyperphenylalaninemia (HPA). Elevated Phe is a marker of this disorder; thus, a commonly used plasma Phe cutoff level for HPA diagnosis is >120 μmol/l (>2 mg/dl) (Blau et al. 2010).
BH4 is an essential cofactor for aromatic amino acid hydroxylases, including phenylalanine, tyrosine, and tryptophan hydroxylases (Arai et al. 1982). Defects in the enzymatic conversion of phenylalanine to tyrosine, tyrosine to l-Dopa, and tryptophan to 5-hydroxytryptophan cause HPA and reduce dopamine and serotonin levels in the central nervous system (Arai et al. 1982).
The classical pathway for the de novo biosynthesis of BH4 from guanosine triphosphate (GTP) requires three enzymes: GTP cyclohydrolase I (GTPCH; EC 3.5.4.16), 6-pyruvoyl-tetrahydropterin synthase (PTPS; EC 4.6.1.10), and sepiapterin reductase (SR; EC 1.1.1.153) (Blau et al. 2010). In addition, dihydropteridine reductase (DHPR; EC 1.6.99.7) and pterin-4a-carbinolamine dehydratase (PCD; EC 4.2.1.96) are critical for the regeneration of BH4.
BH4 deficiency is a severe disease involving progressive neurologic deterioration despite adequate dietary control of blood phenylalanine levels (Arai et al. 1982). Therefore, differential diagnosis to distinguish BH4 deficiencies from PAH gene deficiencies is very important.
In Iran, screening for phenylketonuria (PKU) began as a pilot program in three provinces in 2006 and was expanded nationwide in 2011 under the direction of the Genetics Office of the Ministry of Health.
For a complete study, DHPR enzyme activity measurement and urinary neopterin and biopterin analyses are required to rule out BH4 deficiencies. For this purpose, the National Biochemistry Reference Laboratory (NBRL) at the Pasteur Institute of Iran began to administer tests and serve patients in 2010. This program allows for the early identification and timely intervention to reduce morbidity and mortality rates, thus increasing the chances for healthy patient outcomes.
At present, NBRL is the only referral center in Iran to diagnose these disorders by urinary neopterin and biopterin analyses and DHPR enzyme activity determination. The purpose of this paper is to report on the implementation of these tests in Iran and present results from 4 years (2010–2014) of diagnosing autosomal-recessive BH4 deficiencies at our laboratory.
Materials and Methods
Patients
463 patients diagnosed with HPA (blood phenylalanine >120 μmol/l) and 154 patients with HPA and neurological signs referred by physicians nationwide between 2010 and 2014 were investigated for possible BH4 deficiencies at NBRL.
Selective tests conducted for all patients included blood phenylalanine measurement by high-performance liquid chromatography (HPLC), the DHPR activity test, and urinary neopterin and biopterin analyses by HPLC (Atherton 1989; Ponzone et al. 2004; Blau et al. 2008). Patients were on unrestricted diets for 3 days before sampling. Medications such as multivitamins, BH4, ferrous sulfate, and folic acid were not taken prior to before sampling. Dried blood samples (DBS) on filter paper cards were prepared by blood sampling, as the DHPR enzyme in DBS is stable enough for the sample to be mailed to a central laboratory (Ponzone et al. 2004).
For a complete evaluation to establish the exact nature of the genotype, blood samples from BH4 deficient patients and their parents were sent to the genetic laboratory for molecular analysis. Out of 76 patients, gene mutation analysis was only performed for 44 cases because some patients did not consent to the molecular study.
Neopterin and Biopterin Measurement
Fresh clean-catch urine specimens were used to determine pterin profile levels. All urine samples were diluted 100-fold in normal saline (Ribeiro de Castro et al. 2004). Acid oxidation of reduced pterin was conducted according to the protocol of Blau et al. (2008). A Knauer HPLC system and Waters fluorimeter, equipped with a primary filter exciting at 350 nm and a secondary filter emitting wavelengths at 450 nm, and Tracer Excel 120 reversed-phase columns ODSB 5 μm 25 × 0.46 cm, part number: TR-016345, serial number: NF-31639, (Teknokroma) were used for pterin analysis.
Quality assurance of the neopterin and biopterin measurements was obtained by testing standard solutions and monitoring the inter-assay variation of urine precision control prepared in our lab.
DHPR Activity Assay
Enzyme activity was assayed based on spectrophotometric monitoring of the formation of ferrocytochrome C in a coupled reaction, according to the protocols of Arai et al. (1982) and Blau et al. (2008). Quality assurance of the DHPR assay was obtained by testing and monitoring the inter-assay variation of blood spot elutes from a normal adult and a DHPR-deficient patient.
Genetics
DNA was extracted from 5 ml peripheral blood collected from each patient and their parents. Direct sequencing of PCR products was accomplished using a BigDye Terminator kit (Thermo Fisher Scientific, Life Technologies, USA) according to the manufacturer’s protocol, using an ABI3130XL Genetic Analyzer for mutation detection in the Dr. Zeinali Human Genetics Laboratory, Tehran, Iran. Mutations in genomic DNA in GCH1 (OMIM: 600225), PCBD1 (OMIM: 126090), PTS (OMIM: 612719), and QDPR (OMIM: 612676) genes were studied (http://www.dnalc.org).
The bioinformatics analysis of these genes was performed using Gene Runner version 3.05, and mismatches were compared using an in-house MS Word file containing full details of exons (6 in GCH1, 4 in PCBD1, 6 in PTS, and 7 in QDPR genes), intron numbers, codon numbers, and details of known mutations.
Statistical Analysis
Statistical analysis of the results was performed in Microsoft Excel 2010. Median, minimum, and maximum values were extracted for all variables.
Results
In this cross-sectional study, 617 hyperphenylalaninemic cases were tested for DHPR activity and urinary neopterin and biopterin profiles. According to the data, of 617 HPA patients, 76 cases exhibited BH4 deficiency.
The results, including the median, minimum, and maximum values of the 541 HPA cases, were shown in Table 1. PAH gene mutations were searched in some patients but are not reported in this article.
Table 1.
Biochemical data according to age and phenylalanine level for 541 hyperphenylalaninemic cases
| Phenylalanine 120–600 μmol/L | ||||
|---|---|---|---|---|
| <1 Year | 1–4 Years | 4–10 Years | >10 Years | |
| Phe (B) (μmol/L) | 276 (127; 589) | 262 (126; 583) | 398 (177; 586) | 223 (121; 570) |
| Neo (U) (mmol/mol Creat.) | 3.54 (0.39; 10.10) | 2.42 (0.40; 15.13) | 0.99 (0.76; 2.51) | 0.62 (0.46; 1.15) |
| Bio (U) (mmol/mol Creat.) | 1.65 (0.30; 8.83) | 3.04 (0.63; 9.06) | 1.43 (0.76; 3.77) | 1.68 (0.83; 2.49) |
| DHPR activity (mU/mg Hb) | 2.80 (1.40; 4.70) | 2.80 (1.40; 4.70) | 2.20 (1.80; 4.60) | 2.00 (1.40; 2.60) |
| No. of cases | 149 | 35 | 12 | 4 |
| Phenylalanine 600–1,200 μmol/L | ||||
|---|---|---|---|---|
| <1 Year | 1–4 Years | 4–10 Years | >10 Years | |
| Phe (B) (μmol/L) | 863 (600; 1,199) | 1,020 (600; 1,193) | 1,055 (631; 1,182) | 986 (606; 1,185) |
| Neo (U) (mmol/mol Creat.) | 4.68 (1.25; 9.69) | 2.66 (1.18; 5.43) | 1.77 (0.74; 4.36) | 1.23 (0.64; 2.61) |
| Bio (U) (mmol/mol Creat.) | 3.84 (0.43; 8.55) | 3.52 (0.80; 6.78) | 2.66 (1.43; 5.20) | 2.43 (1.17; 5.19) |
| DHPR activity (mU/mg Hb) | 2.60 (1.60; 4.10) | 2.40 (1.30; 3.50) | 2.00 (1.40; 3.80) | 2.10 (1.60; 2.80) |
| No. of cases | 56 | 30 | 25 | 14 |
| Phenylalanine >1,200 μmol/L | ||||
|---|---|---|---|---|
| <1 Year | 1–4 Years | 4–10 Years | >10 Years | |
| Phe (B) (μmol/L) | 1,778 (1,204; 3,039) | 1,600 (1,206; 2,707) | 1,500 (1,212; 2,100) | 1,551 (1,220; 1,961) |
| Neo (U) (mmol/mol Creat.) | 4.43 (1.47; 21.05) | 3.52 (1.36; 9.54) | 1.95 (0.19; 8.13) | 1.10 (0.41; 2.84) |
| Bio (U) (mmol/mol Creat.) | 4.29 (0.81; 13.50) | 4.06 (0.97; 15.54) | 3.67 (1.00; 10.56) | 1.76 (0.44; 4.80) |
| DHPR activity (mU/mg Hb) | 2.60 (1.40; 4.40) | 2.30 (1.40; 4.90) | 2.00 (1.30; 3.60) | 1.85 (1.30; 3.10) |
| No. of cases | 88 | 45 | 55 | 28 |
Phe (B) phenylalanine in blood, Neo (U) neopterin in urine, Bio (U) biopterin in urine, Creat. urine creatinine, Hb hemoglobin
In the remaining cases, 76 individuals had BH4 deficiencies with the following subtypes: DHPR deficiency (35 patients), PTPS deficiency (37 patients), PCD deficiency (1 patient), and GTPCH deficiency (3 patients). The results, including the median, minimum, and maximum values for laboratory data in these groups are shown in Table 2.
Table 2.
Laboratory data of patients with BH4 deficiency at the time of diagnosis
| BH4 Deficiencies Groups | PTPS Deficiency Group Median (Min; Max) | DHPR Deficiency Group Median (Min; Max) | PCD Deficiency Group Median | GTPCH Deficiency Group Median (Min; Max) | Reference range in normal group |
|---|---|---|---|---|---|
| Phenylalanine (B) (μmol/L) | 864 (60; 2,064) | 264 (49; 1,702) | 1,327 | 1,248 (827; 1,747) | <1 month: 0–124 <16 years: 26–86 ≥16 years: 41–68 |
| Neo (U) (mmol/mol Creat.) | 24.08 (4.09; 57.15) | 2.49 (0.74; 21.82) | 1.52 | <0.2 | 1 day to 10 years: 1.1–4.0 >11 years: 0.2–1.7 |
| Bio (U) (mmol/mol Creat.) | 0.11 (0.02; 0.64) | 8.54 (2.87; 23.03) | – | <0.2 | 1 day to 10 years: 0.5–3 >11 years: 0.5–2.7 |
| % Biopterin | 0.49 (0.06–4.82) | 77.50 (31.46-91.46) | – | – | |
| DHPR activity (mU/mg Hb) | 3.8 (1.70; 4.60) | 0.0 (0.0; 0.0) | – | 4.20 (3.40–4.50) | 1.8–3.8 |
| No. of female cases | 18 | 12 | 1 | 2 | – |
| No. of male cases | 19 | 23 | 0 | 1 | – |
| No. of total (Percentage; %) | 37 (49%) | 35 (46%) | 1 (1%) | 3 (4%) | – |
| Ages | 1 month to 12 years | 1 month to 20 years | 13 years | 5 month to 4 years | – |
Phe (B) phenylalanine in blood, Neo (U) neopterin in urine, Bio (U) biopterin in urine, %Biopterin calculated by ((biopterin/(biopterin + neopterin))*100); Creat. urine creatinine, Hb hemoglobin
Out of 76 patients, 4 patients were Afghan and 6 patients had Arab ethnic origins. All 10 of these patients suffered from PTPS deficiency. For 11 patients with DHPR deficiency and 16 patients with PTPS deficiency, diagnosis occurred prior to one year of age. Consanguineous marriage existed in 64 (84.2%) families. In terms of gender, there was an early equal distribution of PTPS deficiency between females (49%) and males (51%), but in the DHPR deficiency group, the number of males (66%) was twice that of females (34%). Four of the reported cases of DHPR-deficient patients were siblings.
Also, decreases or absences of both neopterin and biopterin were observed in GTPCH deficiency group profile.
In PTPS-deficient patients, neopterin concentrations were significantly increased while biopterin levels were low. The data from this group imply that 37 PTPS-deficient patients (100%) had neopterin >4.0 mmol/mol creatinine, while 35 patients (94.6%) had biopterin ≤0.5 mmol/mol creatinine.
In PCD-deficient patients, elevated neopterin and primapterin levels were observed.
According to the past research, DHPR-deficient subjects showed slightly elevated amounts of biopterin combined with minor increases in neopterin (Blau et al. 2008). In our study, the data showed that 32 DHPR-deficient patients (91.4%) had biopterin >3.0 mmol/mol creatinine. In this group, only 2 cases (5.7%) of 35 DHPR-deficient patients exhibited normal levels of both neopterin and biopterin simultaneously.
Statistical analysis of the phenylalanine results determined that 29 DHPR-deficient patients (82.9%) and 13 PTPS-deficient patients (35.1%) had phenylalanine <600 μmol/l, which demonstrated that most of the BH4-deficient patients have mild HPA.
The genomic structures of identified mutations for 44 cases in GCH1, PCBD1, PTS, and QDPR genes, their inheritance conditions, and other information including biochemical data are shown in detail in Table 3.
Table 3.
Gene mutation analysis results and laboratory data for patients with BH4 deficiencies
| Row | Status | ID number | Date of birth | Age at diagnosis | Gender | Consanguinity | Ethnic origin | Phe (B) at birth μmol/l | Phe (B) at diagnosis μmol/l | Coding DNA reference Sequence | NCBI reference sequence for genes | Exon | Inheritance condition | Neo (U) mmol / mol Creat. | Bio (U) mmol / mol Creat. | % Biopterin | DHPR activity |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | GCTPH Def. | P.1030 | 13.06.2011 | 3.5 years | M | Yes | Fars | 972 | 1,747 | c.551G>A* | NM_000161.2 | 5 | Homozygote | <0.2 | <0.2 | nd | 4.2 |
| 2 | GCTPH Def. | P.2137 | 16.12.2014 | 1.5 months | F | Yes | Fars | 900 | 1,248 | c.551G>A* | NM_000161.2 | 5 | Homozygote | <0.2 | <0.2 | nd | 3.4 |
| 3 | GCTPH Def. | P.2149 | 12.08.2014 | 5 months | F | Yes | Azari | 1,500 | 827 | nd | – | – | – | <0.2 | <0.2 | nd | 4.5 |
| 4 | PCD Def. | P.1017 | 11.08.2000 | 12.5 years | M | Yes | Fars | – | 1,116 | c.313 T>C* | NM_000281.3 | 4 | Homozygote | 1.52 | – | nd | nd |
| 5 | DHPR Def. | P.432 | 28.12.2003 | 7 years | F | No | Azari | – | 220 | c.53G>A | NM_000320.2 | 1 | Homozygote | 1.54 | 9.05 | 85.46 | 0 |
| 6 | DHPR Def. | P.223 | 25.05.2000 | 10 years | M | Yes | Fars | – | – | c.49G>T* | NM_000320.2 | 1 | Homozygote | 1.7 | 5.28 | 75.64 | nd |
| 7 | DHPR Def. | P.1477 | 25.05.2013 | 1.5 months | M | Yes | Fars | 690 | 184 | c.68G>A* | NM_000320.2 | 1 | Homozygote | 3.83 | 3.89 | 50.39 | 0 |
| 8 | DHPR Def. | P.2104 | 09.07.2014 | 6 months | M | Yes | Azari | 635 | 244 | c.190 G>A | NM_000320.2 | 2 | Homozygote | 1.56 | 6.88 | 81.52 | 0 |
| 9 | DHPR Def. | P.800 | 13.07.2011 | 3.5 years | F | Yes | Fars | 360 | 48.6 | c.265G>A* | NM_000320.2 | 3 | Homozygote | 1.79 | 5.08 | 73.94 | 0 |
| 10 | DHPR Def. | P.2195 | 25.11.2008 | 6 years | M | Yes | Talesh | – | 245 | c.266G>A* | NM_000320.2 | 3 | Homozygote | 1.98 | 6.1 | 75.50 | 0 |
| 11 | DHPR Def. | P.1172 | 06.05.2010 | 2.5 years | F | Yes | Fars | 257 | 143 | c.267A>G* | NM_000320.2 | 3 | Homozygote | 3.15 | 11.28 | 78.17 | 0 |
| 12 | DHPR Def. | P.232 | 03.04.2009 | 1.5 years | M | Yes | Lor | – | 228 | c.341C>T* | NM_000320.2 | 4 | Homozygote | 8.47 | 21.53 | 71.77 | 0 |
| 13 | DHPR Def. | P.935 | 12.10.2009 | 2.5 years | M | Yes | Fars | 282 | 714 | c.344C>T* | NM_000320.2 | 4 | Homozygote | 1.76 | 10.13 | 85.20 | 0 |
| 14 | DHPR Def. | P.386 | 29.07.2010 | 4 months | F | Yes | Fars | – | 240 | c.449A>G | NM_000320.2 | 5 | Homozygote | 6.14 | 8.03 | 56.67 | 0 |
| 15 | DHPR Def. | P.892 | 29.01.2004 | 8 years | M | Yes | Fars | – | 506 | c.449A>G | NM_000320.2 | 5 | Homozygote | 0.74 | 6.16 | 89.28 | 0 |
| 16 | DHPR Def. | P.1019 | 22.01.2008 | 5 years | M | No | Azari | 900 | 300 | c.449A>G | NM_000320.2 | 5 | Homozygote | 4.72 | 11.16 | 70.28 | 0 |
| 17 | DHPR Def. | P.1027 | 23.04.1999 | 13 years | M | No | Azari | – | 228 | c.449A>G | NM_000320.2 | 5 | Homozygote | 1.05 | 5 | 82.64 | 0 |
| 18 | DHPR Def. | P.94-89 | 25.12.2005 | 9.5 years | F | Yes | Fars | 440 | 593 | c.470 T>G | NM_000320.2 | 5 | Homozygote | 4.08 | 22.25 | 84.50 | 0 |
| 19 | DHPR Def. | P.94-124 | 03.08.2014 | 1 year | M | Yes | Azari | 600 | 142 | c.472 C>T | NM_000320.2 | 5 | Homozygote | 1.86 | 4.25 | 69.56 | 0 |
| 20 | DHPR Def. | P.1524 | 16.06.2013 | 3 months | F | Yes | Fars | 360 | 551 | c.488G>C* | NM_000320.2 | 5 | Homozygote | 4.53 | 5.26 | 53.73 | 0 |
| 21 | DHPR Def. | P.586 | 03.11.2008 | 2.5 years | M | Yes | Azari | – | 389 | c.217del (c.217delA)* | NM_000320.2 | 7 | Homozygote | 2.64 | 13.1 | 83.23 | 0 |
| 22 | DHPR Def. | P.1117 | 23.12.2011 | 6 months | M | Yes | Fars | 660 | 174 | c.661C>T | NM_000320.2 | 7 | Homozygote | 4.75 | 6.58 | 58.08 | 0 |
| 23 | DHPR Def. | P.366 | 26.02.2002 | 9 years | M | Yes | Azari | – | 525.61 | c.673G>A* | NM_000320.2 | 7 | Homozygote | 2.3 | 18.34 | 88.86 | 0 |
| 24 | DHPR Def. | P.1274 | 31.12.1996 | 16 years | F | Yes | Azari | – | 400.88 | c.673G>A* | NM_000320.2 | 7 | Homozygote | nd | nd | nd | 0 |
| 25 | DHPR Def. | P.1289 | 28.11.2012 | 8 months | M | No | Kord | 312 | 175 | c.710C>T* | NM_000320.2 | 7 | Homozygote | 1.13 | 2.88 | 71.82 | 0 |
| 26 | DHPR Def. | P.94-17 | 29.03.2015 | 1 month | F | Yes | Azari | 600 | 1,702 | c.295 + 1 G>A | NM_000320.2 | – | Homozygote | 10.59 | 4.86 | 31.46 | 0 |
| 27 | DHPR Def. | P.925 | 01.12.2008 | 3 years | F | Yes | Fars | 540 | 534 | nd | – | – | – | 3.33 | 13.9 | 80.67 | 0 |
| 28 | DHPR Def. | P.504 | 05.06.2008 | 3 years | F | Yes | Fars | – | 626 | nd | – | – | – | 3.73 | 12.37 | 76.83 | 0 |
| 29 | DHPR Def. | P.1571 | 10.11.2012 | 1 year | M | Yes | Azari | 312 | 840 | nd | – | – | – | 2.87 | 15.98 | 84.77 | 0 |
| 30 | DHPR Def. | P.582 | 05.08.2008 | 6 years | M | Yes | Azari | – | 241 | nd | – | – | – | 2.34 | 10.83 | 82.23 | 0 |
| 31 | DHPR Def. | P.211 | 27.03.2001 | 11 years | M | Yes | Fars | – | 199 | nd | – | – | – | 1.34 | 6.95 | 83.84 | 0 |
| 32 | DHPR Def. | P.1738 | 14.08.2013 | 6 months | M | Yes | Azari | – | 161 | nd | –– | – | – | 1.13 | 2.87 | 71.75 | 0 |
| 33 | DHPR Def. | P.500 | 09.01.2010 | 15 months | M | No | Baloch | – | 405 | nd | – | – | – | 4.72 | 23.03 | 82.99 | 0 |
| 34 | DHPR Def. | P.1377 | 18.07.2009 | 3.5 years | M | Yes | Lor | – | 240 | nd | – | – | – | 1.19 | 4.86 | 80.33 | 0 |
| 35 | DHPR Def. | P.1383 | 18.05.2008 | 5 years | M | Yes | Bakhtiari | – | 120 | nd | – | – | – | 1.77 | 4.36 | 71.13 | 0 |
| 36 | DHPR Def. | P.1885 | 07.12.2013 | 9 months | M | Yes | Lor | 432 | 387 | nd | – | – | – | 2.7 | 11.21 | 80.59 | 0 |
| 37 | DHPR Def. | P.1948 | 08.09.1994 | 20 years | M | Yes | Tork | – | 283 | nd | – | – | – | 1.59 | 17.02 | 91.46 | 0 |
| 38 | DHPR Def. | P.94.145 | 14.06.2014 | 2 months | F | Yes | Fars | 240 | 1,503 | nd | – | – | – | 11.04 | 10.87 | 49.61 | 0 |
| 39 | DHPR Def. | P.1917 | 22.05.2014 | 1.5 months | F | Yes | Fars | 570 | 1,020 | nd | – | – | – | 21.82 | 11.38 | 34.28 | 0 |
| 40 | PTPS Def. | P.1160 | 14.04.2011 | 1.5 years | M | No | Afghan | – | 940 | c.155A>G | NM_000317.2 | 2 | Homozygote | 48.18 | 0.35 | 0.72 | nd |
| 41 | PTPS Def. | P.247 | 21.06.2008 | 2.5 years | F | Yes | Fars | – | 672 | c.155A>G | NM_000317.2 | 2 | Homozygote | 42.9 | 0.55 | 1.27 | nd |
| 42 | PTPS Def. | P.246 | 21.01.2008 | 2.5 years | F | Yes | Fars | – | 766.02 | c.155A>G | NM_000317.2 | 2 | Homozygote | 51.49 | 0.44 | 0.85 | nd |
| 43 | PTPS Def. | P.2030 | 27.08.2014 | 4.5 months | F | Yes | Azari | 630 | 807 | c.199 C>T | NM_000317.2 | 2 | Homozygote | 36.57 | 0.23 | 0.63 | 4.3 |
| 44 | PTPS Def. | P.1816 | 18.04.2011 | 3 years | M | Yes | Fars | 180 | 124 | c.259 C>T | NM_000317.2 | 5 | Homozygote | 7.65 | 0.03 | 0.39 | 1.9 |
| 45 | PTPS Def. | P.227 | 30.01.2009 | 6 months | F | Yes | Kord | – | 947 | c.281A>T* | NM_000317.2 | 5 | Homozygote | 54.23 | 0.25 | 0.46 | nd |
| 46 | PTPS Def. | P.238 | 18.08.2009 | 11.5 months | M | Yes | Kord | – | 800.55 | c.281A>T* | NM_000317.2 | 5 | Homozygote | 13.31 | 0.02 | 0.15 | nd |
| 47 | PTPS Def. | P.1157 | 26.04.2012 | 4.5 months | F | No | Fars | 794 | 1,353 | c.297C>A & c.84-3C>G | NM_000317.2 | 5 | Compound Heterozygote | 35.5 | 0.25 | 0.70 | nd |
| 48 | PTPS Def. | P.2001 | 04.03.2015 | 7 months | F | Yes | Fars | 1,440 | 1,852 | c.317 C>T | NM_000317.2 | 6 | Homozygote | 34.9 | 0.08 | 0.23 | 2.6 |
| 49 | PTPS Def. | P.2194 | 17.01.2015 | 1.5 months | F | No | Azari | 1,560 | 1,305 | c.317 C>T | NM_000317.2 | 6 | Homozygote | 35.99 | 0.15 | 0.42 | 4.6 |
| 50 | PTPS Def. | P.207 | 05.03.2001 | 12 years | F | Yes | Azari | – | 2,064 | c.331G>A* | NM_000317.2 | 6 | Homozygote | 11.87 | 0.04 | 0.34 | nd |
| 51 | PTPS Def. | P.1551 | 04.08.2013 | 2 months | M | Yes | Afghan | 841 | 596 | c.351C>A* | NM_000317.2 | 6 | Homozygote | 12.57 | 0.16 | 1.26 | nd |
| 52 | PTPS Def. | P.236 | 10.05.2005 | 5 years | M | Yes | Arab | – | 480 | c.373A>T | NM_000317.2 | 6 | Homozygote | 31.72 | 0.04 | 0.13 | nd |
| 53 | PTPS Def. | P.94-26 | 19.12.2014 | 6 months | F | Yes | Arab | 1,230 | 1,616 | c.373A>T | NM_000317.2 | 6 | Homozygote | 57.15 | 0.19 | 0.33 | 3.8 |
| 54 | PTPS Def. | P.1124 | 06.11.2008 | 4 years | M | Yes | Arab | – | 420 | c.373A>T | NM_000317.2 | 6 | Homozygote | 24.08 | 0.07 | 0.29 | nd |
| 55 | PTPS Def. | P.1344 | 15.09.2011 | 1.5 years | M | Yes | Fars | 660 | 1,331 | c.373A>T | NM_000317.2 | 6 | Homozygote | 22.06 | 0.11 | 0.50 | nd |
| 56 | PTPS Def. | P.1268 | 08.12.2012 | 1 month | F | Yes | Azari | 1,020 | 1,241 | c.400G>A* | NM_000317.2 | 6 | Homozygote | 33.13 | 0.02 | 0.06 | nd |
| 57 | PTPS Def. | P.229 | 03.07.2009 | 1 year | M | Yes | Kord | 480 | 90 | c.84-3C>G | NM_000317.2 | – | Homozygote | 6.31 | 0.32 | 4.83 | nd |
| 58 | PTPS Carrier | P.1918 | 05.06.2014 | 1 month | F | Yes | Fars | 270 | 244 | c.163 + 2 T>C* | NM_000317.2 | – | Heterozygote | 10.01 | 3.14 | 23.88 | 2.7 |
| 59 | PTPS Def. | P.1122 | 19.02.2012 | 6.5 months | M | Yes | Arab | 1,920 | 1,200 | c.164.36A>G* | NM_000317.2 | – | Homozygote | 48.85 | 0.04 | 0.08 | nd |
| 60 | Probably PTPS Def. | P.878 | 06.12.2010 | 1 year | M | Yes | Kord | – | 60 | nd | – | – | – | 11.1 | 0.12 | 1.07 | nd |
| 61 | Probabely PTPS Def. | P.1105 | 12.04.2012 | 4 months | F | Yes | Fars | 864 | 894 | nd | – | – | – | 25.83 | 0.08 | 0.31 | nd |
| 62 | Probabely PTPS Def. | P.622 | 01.10.2010 | 9 months | M | No | Fars | – | – | nd | – | – | – | 20.4 | 0.06 | 0.29 | nd |
| 63 | Probabely PTPS Def. | P.1213 | 23.04.2012 | 1.5 years | M | Yes | Fars | – | 1,410 | nd | – | – | – | 29.48 | 0.15 | 0.51 | 4.1 |
| 64 | Probabely PTPS Def. | P.260 | 02.12.2007 | 5 years | F | No | Fars | – | 791 | nd | – | – | – | 16.27 | 0.1 | 0.61 | nd |
| 65 | Probabely PTPS Def. | P.563 | 26.08.2007 | 3 years | M | Yes | Fars | – | 1,129 | nd | – | – | – | 18.06 | 0.1 | 0.55 | nd |
| 66 | Probabely PTPS Def. | P.596 | 31.10.2005 | 5 years | M | Yes | Azari | – | 426 | nd | – | – | – | 4.09 | 0.08 | 1.92 | nd |
| 67 | Probabely PTPS Def. | P.1141 | 16.08.2006 | 7 years | M | Yes | Kord | – | 1,156 | nd | – | – | – | 22.82 | 0.11 | 0.48 | nd |
| 68 | Probabely PTPS Def. | P.1375 | 23.08.2006 | 6.5 years | F | Yes | Arab | – | 540 | nd | – | – | – | 13.81 | 0.09 | 0.65 | nd |
| 69 | Probabely PTPS Def. | P.357 | 07.06.2010 | 4.5 months | F | Yes | Azari | – | 1,890 | nd | – | – | – | 55.08 | 0.64 | 1.15 | nd |
| 70 | Probabely PTPS Def. | P.1591 | 20.09.2012 | 13 months | F | Yes | Azari | 510 | 1,080 | nd | – | – | – | 16.05 | 0.07 | 0.43 | 1.8 |
| 71 | Probabely PTPS Def. | P.1717 | 15.08.2013 | 5 months | F | No | Afghan | – | 1,573 | nd | – | – | – | 30.23 | 0.21 | 0.69 | 1.7 |
| 72 | Probabely PTPS Def. | P.1736 | 23.03.2009 | 5 years | M | Yes | Azari | 1,320 | 834 | nd | – | – | – | 16.62 | 0.2 | 1.19 | 1.8 |
| 73 | Probabely PTPS Def. | P.1813 | 27.11.2011 | 2 years | F | Yes | Fars | – | 1,251 | nd | – | – | – | 19.09 | 0.16 | 0.83 | nd |
| 74 | Probabely PTPS Def. | P.94-128 | 23.08.2014 | 1 year | M | Yes | Afghan | – | 585 | nd | – | – | – | 22.43 | 0.1 | 0.44 | 4.3 |
| 75 | Probabely PTPS Def. | P.94-48 | 30.01.2014 | 1.5 years | M | Yes | Arab | – | 579 | nd | – | – | – | 24.63 | 0.11 | 0.44 | 3.9 |
| 76 | Probabely PTPS Carrier | P.94-95 | 11.06.2015 | 1 month | M | No | Azari | 360 | 488 | nd | – | – | – | 45.62 | 2.03 | 4.26 | 4 |
Phe (B) phenylalanine in blood, Neo (U) neopterin in urine, Bio (U) biopterin in urine, %Biopterin calculated by ((biopterin/(biopterin + neopterin))*100), nd not detected
In our study, we found that mutations more frequently involved DHPR (46%) and PTPS (49%) than GTPCH (4%) and PCD (1%) (Table 3). All BH4-deficient patients showed homozygous mutations, except one patient in the PTPS deficiency group, who had c.297C>A and c.84-3C>G in a compound heterozygous form.
Most detected mutations had been previously reported (http://www.biopku.org/BioPKU_DatabasesBIOMDB.asp), but 18 missense mutations (16 in exons and 2 in introns) and 1 deletion mutation were novel and had not been registered in the BioPKU_Databases BIOMDB yet. They are demonstrated by asterisks in Table 3 and included: c.163 + 2 T>C, c.217del(c.217delA), c.265G>A, c.266G>A, c.267A>G, c.673G>A, c.710C>T, c.49G>T, c.68G>A, c.488G>C, and c.344C>T in QDPR gene;c.281A>T,c.164.36A>G,c.331G>A,c.400G>A,c.351C>A, and c.163 + 2 T>C in PTS gene; c.313 T>C in PCBD1 gene; c.551G>A in GCH1 gene.
Moreover, the diagnosis for one of the DHPR-deficient patients with DHPR activity equal to zero was performed by the University Children’s Hospital of Zürich, Switzerland. All exons of the QDPR gene plus flanking intronic regions, and the complete coding sequence of the cDNA, were tested in this case. The new homozygous alteration c.267A>G, which seems to be synonymous for coding on the protein level (p. Gly89Gly) was found. The program used was “Human Splicing Finder,” version 2.4.1 (http://www.umd.be/HSF/). Mutations in c.265G>A which coded (p.Gly89Arg) on the protein level and c.266G>A, coding (p.Gly89Gln) on the protein level, were observed in two other DHPR-deficient patients. All three mutations are novel on Cd89 DHPR gene. Phenylalanine levels were <250 μmol/: 49 μmol/l for patient with mutation in c.265G>A, 245 μmol/l for patient with mutation in c.266G>A, and 143 μmol/l for patient with mutation in c.267G>G.
Discussion
BH4 deficiencies are a very heterogeneous group of diseases (Blau et al. 2001). Every newborn with even slight but persistent HPA should be tested for BH4 deficiency. Such tests have been introduced in many developed countries, but even today, older children are more commonly diagnosed after the appearance of clinical symptoms, such as hypotonia of the trunk, hypertonia of the extremities, and often, myoclonic seizures unresponsive to a low-phenylalanine diet.
Because classical BH4 deficiencies are a group of diseases that can be detected but not identified through neonatal mass screening for HPA, selective screening for a BH4 deficiency is essential for every newborn with slightly elevated phenylalanine levels (Dhondt 1991).
Our findings show that there is no predominant mutation and that the majority of these mutations are isolated in families and scattered throughout the genes of the Iranian population. This finding aligns with a prior Iranian study about the QDPR gene (Foroozani et al. 2015). Furthermore, eight of the eleven new mutations reported in this article on the QDPR gene were previously reported by Foroozani et al. (2015).
It is interesting that in the DHPR deficiency group, the new homozygous alteration c.267A>G in the QDPR gene reported in this paper as a novel mutation, which seems to be synonymous coding on the protein level (p.Gly89Gly), is not an SNP. This mutation generates a new 9G8 (or SR-protein SFRS7) exon-splice-enhancer, which might alter pre-mRNA splicing.
The observation of two other new mutation on codon 89 (c.265G>A and c.266G>A) is a more interesting note that should be reported about Iranian DHPR-deficient patients. All mutations on this codon were novel, and all three patients had mild HPA. Therefore it will be interesting if a phenotype–genotype relation study were carried out.
In the PTPS deficiency group, there were no genetic study data or enzyme activity to confirm disease existence for 17 (46%) patients. According to biochemical findings and clinical signs, they were suspected of having PTPS deficiency. Despite the lack of confirmation for these patients, according to physicians’ comments, they were treated with BH4 tablets.
For a more complete diagnosis it is necessary to measure PTPS activity and assess 5-hydroxyindoleacetic acid, homovanillic acid, and 5-methyl tetrahydrofolic acid in the cerebrospinal fluid. The results in this article show gaps in the prevention and control programs for the diseases. These gaps can often be eliminated by using existing capacities in a standardized and systematic approach. If all newborns with HPA are screened to rule out BH4 deficiencies within the first month of life, we would be able to report a precise incidence of BH4 deficiencies per 100,000 live births in Iran in the future.
Under the present circumstances, BH4-deficient newborns are benefiting from the availability of these tests in the context of a screening program in Iran, because they have the chance to be diagnosed and receive necessary care before clinical damage occurs.
Acknowledgement
We are grateful to the many colleagues in contributing medical centers, the patients’ families, and their physicians for sending specimens. We would also like to give special thanks to Prof. Dr. Nenad Blau, Prof. Dr. Jörn Oliver Sass, Corinne Britschgi, Alessio Cremonesi, and Anahita Rassi for their helpful recommendations and efforts.
Compliance with Ethics Guidelines
Consortium
Soghra Khani, Rogiyeh Mirzazadeh, Sedigheh Sadeghi, Somayeh Mahmoudi Baram, Elham Farhangara, Arezou Asgari, Rayhaneh Hasanzaeh, Mina Barzegari, Parastoo Bayat, Hamid Mohammadaliha, Parinaz Saeedi, Robabeh Ahadi, Ghazaleh Dadashizadeh, Saeedeh Saeedi, Sarah Azadmehr, Tina Shirzad, Leali Rejali, and Mahbobeh Masoodifar.
Conflict of Interest
Shohreh Khatami, Soghra Rouhi Dehnabeh, Sirous Zeinali, Beat Thöny, Mohammadreza Alaei, Shadab Salehpour, Aria Setoodeh, Farzaneh Rohani, Fatemeh Hajivalizadeh, and Ashraf Samavat declare that they have no conflict of interest.
Informed Consent
The project was supported financially with research grants for implemented programs by the Ministry of Health of Iran. Informed consent was obtained from all patients for inclusion in the study.
Animal Rights
This article does not reference any studies with animal subjects performed by any of the authors.
Details of the Contributions of Individual Authors
Shohreh Khatami, as member of the PKU national scientific committee, contributed to the planning, conducting, and reporting of the BH4 screening tests described in the article.
Soghra Rouhi contributed to the conception and design of the DHPR test and peterin analysis setup and to drafting the article.
Mohammadreza Alaei, Shadab Salehpour, Aria Sotoudeh, and Farzaneh Rohani contributed as members of the PKU national scientific committee and introduced patients for sample gathering.
Sirous Zeinali, Sarah Azadmeh, Tina Shirzad, Leyli Rejali, Mahbobeh Masoodifarand, and Beat Thöny contributed to the genetics study.
Soghra Khani, Rogiyeh Mirzazadeh, Sedigheh Sadeghi, Somayeh Mahmoudi Baram, Elham Farhangara, Arezu Asgari, Ghazaleh Dadashizadeh, Rayhaneh Hasanzaeh, Mina Barzegari, Parinaz Saeedi, Parastoo Bayat, Robabeh Ahadi, Hamid Mohammadaliha, and Saeedeh Saeedi contributed to the setup and performing of the tests.
Ashraf Samavat (Head of the Genetics Office, Ministry of Health of Iran) and Fatemeh Hajivalizadeh contributed to the design of the PKU screening program in Iran and to revising the article critically for important intellectual content.
Author Serving as Guarantor for the Article
Dr. Shohreh Khatami
Competing Interests Statement
None
Contributor Information
Shohreh Khatami, Email: sh-khatami@pasteur.ac.ir.
Soghra Rouhi Dehnabeh, Email: SRD@pasteur.ac.ir.
Collaborators: Matthias R. Baumgartner, Marc Patterson, Shamima Rahman, Verena Peters, Eva Morava, and Johannes Zschocke
References
- Arai N, Narisawa K, Hayakawa H, et al. Hyperphenylalaninemia due to dihydropteridine reductase deficiency: diagnosis by enzyme assays on dried blood spots. Pediatrics. 1982;70:426–430. [PubMed] [Google Scholar]
- Atherton ND. HPLC measurement of phenylalanine by direct injection of plasma onto an internal-surface reversed-phase silica support. Clin Chem. 1989;35(6):975–978. [PubMed] [Google Scholar]
- BIOMDB: Database of Mutations Causing Tetrahydrobiopterin Deficiencies (database online) (http://www.biopku.org/BioPKU_DatabasesBIOMDB.asp) curated by N. Blau, B. Thöny
- Blau N, Thöny B, Cotton RGH. Disorders of tetrahydrobiopterin and related biogenic amines. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Vogelstein B, editors. The metabolic and molecular bases of inherited disease. 8. New York: McGraw-Hill; 2001. pp. 1725–1776. [Google Scholar]
- Blau N, Duran M, Gibson KM. Laboratory guide to the methods in biochemical genetics techniques. Heidelberg: Springer; 2008. pp. 696–699. [Google Scholar]
- Blau N, Burton BK, Thöny B, et al. Phenylketonuria and BH4 deficiencies. 1. Bremen: UNI-MED; 2010. [Google Scholar]
- Dhondt JL. Strategy for the screening of tetrahydrobiopterin deficiency among hyperphenylalaninaemic patients: 15-years experience. J Inherit Metab Dis. 1991;14:117–127. doi: 10.1007/BF01800581. [DOI] [PubMed] [Google Scholar]
- Foroozani H, Abiri M, Salehpour S, et al. Molecular characterization of QDPR gene in Iranian families with BH4 deficiency: reporting novel and recurrent mutations. JIMD Rep. 2015;21:123–128. doi: 10.1007/8904_2015_441. [DOI] [PMC free article] [PubMed] [Google Scholar]
- http://www.dnalc.org/view/15479-Sanger-method-of-DNA-sequencing-3D-animation-with-narration.html
- Ponzone A, Spada M, Ferraris S, et al. Dihydropteridine reductase deficiency in man: from biology to treatment. Med Res Rev. 2004;24(2):127–150. doi: 10.1002/med.10055. [DOI] [PubMed] [Google Scholar]
- Ribeiro de Castro M, Seno Di Marco G, Yuri Arita D, et al. Urinary neopterin quantification by reverse-phase high-performance liquid chromatography with ultraviolet detection. J Biochem Biophys Methods. 2004;59(3):275–283. doi: 10.1016/j.jbbm.2004.03.004. [DOI] [PubMed] [Google Scholar]