The T/G 13915 variant upstream of the lactase gene (LCT) is the founder allele of lactase persistence in an urban Saudi population.

BACKGROUND: The prevalence of lactase persistence is high in Saudi Arabia. OBJECTIVE: To identify a DNA variant for the lactase persistence/non-persistence trait in adult Arabs in Saudi Arabia. METHODS: We sequenced DNA from 432 anonymous neonatal blood donors from five different regions of Saudi Arabia to cover the 400 bp region surrounding the previously identified lactase persistence/non-persistence variant C/T-13910 residing in intron 13 of the MCM6 gene. RESULTS: Two anonymous blood donors carried the C/T-13910 genotype. One variant, T/G -13915, residing 5 bp upstream of the C/T-13910 variant, was present in 332 of 432 (76.9%) of the neonatal samples, compatible with previous prevalence figures of lactase persistence in urban Saudi populations. Determination of disaccharidase activities in 25 intestinal biopsy samples showed a highly significant correlation between lactase activity and the T/G-13915 genotypes (p<0.001; Fisher exact test) as well as between the L:S ratio and the aforementioned genotypes (p<0.001; Fisher exact test). CONCLUSION: The T/G-13915 variant is the founder mutation of lactase persistence in an urban Saudi population. The results obtained here have implications for genetic testing of adult-type hypolactasia and to analysis of human evolution, the origin of cattle domestication and migrations of the populations in the Arabian peninsula.

LC-MS/MS determination of dibasic amino acids for the diagnosis of cystinuria. Application in a family affected by a novel splice-acceptor site mutation in the SLC7A9 gene.

Cystinuria is an autosomal recessive disorder caused by defective transport of cystine and the dibasic amino acids ornithine, lysine and arginine across cell membranes. Poor solubility of cystine in urine leads to kidney stones and associated symptoms and complications. Mutations of genes SLC3A1 and SLC7A9 encoding for amino acid transport systems are responsible for different types of cystinuria. In this study we describe a new LC-MS/MS assay for these amino acids in urine. Moreover, we report a novel splice-acceptor site mutation in the SLC7A9 gene that we believe is the cause of the phenotype observed in four siblings from a first-cousin marriage. Into the wells of a 96-well microtitre plate, 10 microl of urine was mixed with 90 microl of a solution containing [(2)H4]cystine, [(2)H2]ornithine, [(13)C,(2)H4]arginine and [(2)H5]glutamine that was used as an internal standard for lysine. Chromatographic separation was achieved isocratically and detection was in the selected-reaction monitoring mode. The injection-to-injection time was 8 min. Calibration curves were linear up to 1000 micromol/L. Intra-day (n = 10) and inter-day (n = 6) variations (750 and 10 micromol/L) were less than 11.4%. Urine samples from healthy individuals (n = 135) were analysed and age-matched reference ranges were generated. The method was applied retrospectively and prospectively to analyse samples (n = 13) from nine cystinuria patients. The mutation reported here was not found in 100 controls with similar ethnicity to the studied family and is believed to have consequences for the transcribed mature RNA and protein structure and function.

Newborn screening: experiences in the Middle East and North Africa.

This review presents the current experiences with newborn screening in the Middle East and North Africa region. The population in the region is about 400 million, with high birth rate and an estimated 10 million newborns per year. The majority of the population is of the Islamic faith and mostly Arab. The population is characterized by a high consanguinity (25-70%) and a high percentage of first-cousin marriages. Haemoglobin disorders, inherited metabolic disorders, neurogenetic disorders and birth defects are relatively common among the population. There is a rather slow progress in developing and implementing preventive genetic programmes owing to legal, cultural, political and financial issues. Although research spending is rather soft in the region, there are numerous pilot studies that highlighted the high incidence of genetic defects and the need for newborn screening programmes. Currently, there are only four countries that are executing national newborn screening but they vary from one disease to 23 and coverage is not complete. The region needs to take big steps towards developing national strategies for prevention and should learn from experiences of regional and international screening programmes.

Quantification of N-acetylaspartic acid in urine by LC-MS/MS for the diagnosis of Canavan disease.

Canavan disease is an autosomal recessive leukodystrophy characterized by excessive excretion of N-acetylaspartic acid (NAA) in urine. The disease is caused by deficiency of aspartoacylase, the enzyme responsible for the hydrolysis of NAA into acetate and l-aspartate. Patients, who are often asymptomatic in their early months, show a wide spectrum of clinical presentation thereafter that includes macrocephaly, poor head control, seizures, abnormal muscle tone, optic atrophy, significant developmental delay and death. In this work, we describe a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of NAA in urine. The internal standard d3-NAA was added to untreated urine and the mixture was injected into the LC-MS/MS system operated in the negative ion mode. Detection was achieved in multiple reaction monitoring (MRM) mode by monitoring m/z 174 --> 88, 174 --> 130 and 174 --> 58 for NAA and 177 --> 89 for the internal standard. Separation was carried out on a C8 column (2.1 x 150 mm) using a mixture of acetonitrile and water (1:1 v/v) containing 0.05% formic acid at a flow rate of 0.25 ml/min. NAA was eluted at 1.6 min and the run time was approximately 2 min. Using spiked urine, the assay was linear up to 2 mmol/L with limit of quantification at 1 micromol/L (S/N = 12). NAA in patients' urine (n = 17) ranged between 366 and 21,235 mmol/mol creatinine compared to controls of <39 mmol/mol creatinine (n = 159). This LC-MS/MS method for NAA as described involved no extraction and no derivatization, showed no interference, and gave excellent recovery with low variability and short analytical time.

Total truncation of the molybdopterin/dimerization domains of SUOX protein in an Arab family with isolated sulfite oxidase deficiency.

We ascertained a patient with the full-blown phenotype of isolated sulfite oxidase deficiency in a consanguineous Arab family. The proband's phenotype included the presence of intractable seizures in the neonatal period, some dysmorphic features, neuroradiologic findings reminiscent of hypoxic ischemic encephalopathy and rapidly progressive brain destruction leading to severe neurodevelopmental impairment. Biochemically, the patient excreted a large amount of S-sulfocysteine with normal amounts of xanthene and hypoxanthine and had normal plasma uric acid, which was consistent with isolated sulfite oxidase deficiency. We report the identification of the first Arab mutation in SUOX, the gene for sulfite oxidase enzyme, in the ascertained family. The newly identified Arab mutation in the SUOX gene (a single nucleotide deletion, del G1244) is predicted to cause a frame shift at amino acid 117 of the translated protein with the generation of a stop codon and total truncation of the molybdo-pterin- and the dimerizing-domain(s) of SUOX protein expressed from the mutant allele. The identification of this new Arab SUOX mutation should facilitate pre-implantation genetic diagnosis and selection of unaffected embryos for future pregnancy in the ascertained family with the mutation and related families with the same mutation.

Identification of a common novel mutation in Saudi patients with argininosuccinic aciduria.

We have identified a common novel mutation (Q354X) in the argininosuccinate lyase (ASL) gene in Saudi patients with argininosuccinic aciduria (ASAuria; McKusick 207900). The two index patients were siblings, had a neonatal onset of the disease and were diagnosed based on the clinical presentation and confirmed by analysis of their dried blood spots (DBS) by tandem mass spectrometry (MS/MS). The ASL gene was then analysed by direct sequencing. A further 28 patients with a confirmed diagnosis of ASAuria based on MS/MS of their DBS were tested by sequencing for the presence of the Q354X mutation. This mutation was found in 14 out of the 28 patients (50%) tested. Our work indicates that the Q354X allele is common, may account for 50% of the abnormal ASL genes in the Saudi population, and is likely to be associated with the neonatal form of the disease. We recommend that all patients diagnosed with ASAuria in Saudi Arabia or of Arab origin be tested for this mutation and for Q116X, which has been described previously. In addition, further analysis is needed to identify other underlying disease mutations for ASAuria in the Saudi population.

Identification of two novel mutations in OCTN2 from two Saudi patients with systemic carnitine deficiency.

Systemic carnitine deficiency (CDSP) (McKusick 212140) is a rare autosomal recessive disease caused by defective plasma membrane uptake of carnitine. The disease is characterized by Reye syndrome, progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. CDSP is a treatable disease provided an early diagnosis is made and prompt treatment with L-carnitine is initiated. The biochemical diagnosis of the disease is based on the findings of very low plasma and tissue carnitine concentrations. Recently, a human gene, SLC22A5, encoding a sodium-dependent high-affinity carnitine transporter OCTN2 was cloned from human kidney and shown to be mutated in systemic carnitine deficiency. Here we report two unrelated Saudi CDSP patients who were detected by tandem mass spectrometric analysis (MS/MS) of blood spots. Studies in skin fibroblasts from the two patients showed a severely reduced carnitine uptake. Subsequent molecular studies led to the identification of two novel missense mutations in the OCTN2 gene in the two patients.

Carnitine palmityl transferase I deficiency.

Carnitine palmityl transferase I is the key enzyme in the carnitine dependent transport of long chain fatty acids across the mitochondrial inner membrane and its deficiency results in a decrease rate of fatty acids beta-oxidation with decreased energy production. We reported a family of 3 affected siblings who are the product of a first degree cousin marriage. The first 2 presented with typical Reye-like syndrome with unconsciousness, hepatomegaly, hypoglycemia, hyperammonemia and very high liver enzymes. Liver biopsy showed steatosis. On screening of the complete family, the 3rd sibling was found to have hepatomegaly. The 3 siblings showed an acyl carnitine profile with very high free carnitine with almost absent long chain acyl carnitines, suggestive of carnitine palmityl transferase I deficiency. This was confirmed by enzyme analyses in fibroblast cultures. These patients were effectively treated with a diet high in carbohydrate, low in long chain fatty acids with medium chain triglycerides. In conclusion, carnitine palmityl transferase I deficiency is an important cause of Reye-like syndrome, which may be treated easily with very good results if detected early in life.

Determination of L-pipecolic acid in plasma using chiral liquid chromatography-electrospray tandem mass spectrometry.

BACKGROUND: L-pipecolic acid (L-PA), an important biochemical marker for the diagnosis of peroxisomal disorders, is usually determined as the racemate. We developed a chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of L-PA in plasma. METHODS: We used a narrow bore chiral macrocyclic glycopeptide teicoplanin column for the enantioseparation of D-pipecolic acid (D-PA) and L-PA and interfaced the column directly to the electrospray source of a tandem mass spectrometer. We used phenylalanine-d5 as internal standard added to 50 microL of plasma followed by deproteinization, evaporation, and injection. The analysis was performed in the selected-reaction monitoring mode using two transitions: m/z 130-->m/z 84 for PA, and m/z 171-->m/z 125 for phenylalanine-d5. L-PA eluted at 7 min, and D-PA eluted at 11.7 min, whereas phenylalanine-d5 eluted at 6 min. The turnaround time for the assay was 20 min. RESULTS: Linear calibration curves were obtained in the range of 0.5-80 micromol/L. At a plasma concentration of 1.0 micromol/L, the signal-to-noise ratio was 50:1. The intra- and interassay variations were 3.1-7.9% and 5.7-13%, respectively, at concentrations of 1-50 micromol/L. Mean recoveries of L-PA added to plasma were 95% (5 micromol/L) and 102% (50 micromol/L). The method clearly distinguished between healthy individuals and peroxisomal disease patients. CONCLUSIONS: The novel LC-MS/MS method is simple, rapid, and stereoselective, and uses only 50 microL of plasma, no derivatizing reagents, and a commercially available internal standard. Sample preparation is not complex and is faster than for all other methods.

Clinical applications of tandem mass spectrometry: ten years of diagnosis and screening for inherited metabolic diseases.

This paper reviews the clinical applications of tandem mass spectrometry (MS-MS) in diagnosis and screening for inherited metabolic diseases in the last 10 years. The broad-spectrum of diseases covered, specificity, ease of sample preparation, and high throughput provided by the MS-MS technology has led to the development of multi-disorder newborn screening programs in many countries for amino acid disorders, organic acidemias, and fatty acid oxidation defects. Issues related to sample acquisition, sample preparation, quantification of metabolites, and validation are discussed. Our current experience with the technique in screening is presented. The application of MS-MS in selective screening has revolutionized the field and made a major impact on the detection of certain disease classes such as the fatty acid oxidation defects. New specific and rapid MS-MS and LC-MS-MS methods for highly polar small molecules are supplementing or replacing some of the classical GC-MS methods for a multitude of metabolites and disorders. New exciting applications are appearing in fields of prenatal, postnatal, and even postmortem diagnosis. Examples for pitfalls in the technique are also presented.

A severe genotype with favourable outcome in very long chain acyl-CoA dehydrogenase deficiency.

A patient with very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is reported. He had a severe neonatal presentation and cardiomyopathy. He was found to be homozygous for a severe mutation with no residual enzyme activity. Tandem mass spectrometry on dried blood spots revealed increased long chain acylcarnitines. VLCAD enzyme activity was severely decreased to 2% of control levels. Dietary management consisted of skimmed milk supplemented with medium chain triglycerides and L-carnitine. Outcome was good and there was no acute recurrence.

Chiral liquid chromatography tandem mass spectrometry in the determination of the configuration of 2-hydroxyglutaric acid in urine.

D-2-Hydroxyglutaric aciduria and L-2-hydroxyglutaric aciduria are two distinct inherited metabolic diseases. The accurate diagnosis of the exact disorder relies on the determination of the configuration of the enantiomers, either D-2-hydroxyglutaric acid or L-2-hydroxyglutaric acid excreted in excess in urine of patients. The enantiomeric chiral separation of 2-hydroxyglutaric acid was achieved using a ristocetin A glycopeptide antibiotic silica gel bonded column. The chiral column was interfaced with a tandem mass spectrometer for the purpose of specifically detecting the eluting 2-hydroxyglutaric acid. Tandem mass spectrometry was employed using an electrospray ion source in the negative ion mode. Three parent-to-daughter transitions under collision-induced dissociation conditions were used to detect only 2-hydroxyglutaric acid. The two forms of the compound were satisfactorily separated with almost baseline resolution at 4.95 and 5.5 min. Three known patients with 2-hydroxyglutaric aciduria were identified to have L-2-hydroxyglutaric aciduria. The method is simple, selective, rapid, and free from interference.

Glutaric aciduria type II: observations in seven patients with neonatal- and late-onset disease.

The clinical, biochemical, and neuroradiologic findings and clinical follow-up of seven patients with glutaric aciduria type II are reported. Three phenotypes of the disease are encountered: neonatal-onset form with congenital anomalies (two patients) or without congenital anomalies (three patients) and late-onset form (two patients). The neonatal-onset form presents as an overwhelming illness, with severe hypoglycemia and metabolic acidosis leading to rapid death. Frequently it is associated with perinatal energy deprivation, a neonate with low birth weight and prematurity. The late-onset form presents with intermittent episodes of vomiting, hypoglycemia, and acidosis especially after meals rich in fat and/or proteins. All parents are consanguineous and have a first- or second-degree relationship. Initially, in the two phenotypes with neonatal onset and during crisis in the late-onset phenotype, routine laboratory evaluation showed severe metabolic acidosis, with an increased anion gap, hypoglycemia without ketonuria, and disturbed liver function tests. In the majority of patients with neonatal-onset forms, the kidneys, liver, and at times the spleen are enlarged with an increased echogenic pattern; however, no hepatic or renal cysts are detected. Cardiomegaly is observed in most patients. The diagnosis can be easily and rapidly reached through tandem mass spectrometry study of the blood and can further be confirmed by gas chromatography/mass spectrometry analysis of the urine organic acids. In this report, the magnetic resonance imaging/computed tomography brain studies showed brain atrophy, white matter disease, and in one patient, fluid-filled cavities in the periventricular area and putamina. Fluorine-18-labeled 2-fluoro-2-deoxyglucose positron emission tomographic (FDG PET) brain studies in two patients with late-onset disease showed slightly decreased activity in the cerebral cortex in one and in the caudate nuclei in the other. Brain FDG PET scan and magnetic resonance spectroscopy were normal in one patient with neonatal-onset disease. All patients were treated with a diet low in fat and protein, oral riboflavin, and carnitine. The results were promising for the late-onset disease. Intravenous carnitine gave rewarding results in one patient with neonatal-onset disease.

Clinical, biochemical, and molecular characterization of patients with glutathione synthetase deficiency.

Pyroglutamic aciduria (5-oxoprolinuria) is a rare autosomal recessive disorder caused by either glutathione synthetase deficiency (GSSD) or 5-oxoprolinase deficiency. GSSD results in low glutathione levels in erythrocytes and may present with hemolytic anemia alone or together with pyroglutamic aciduria, metabolic acidosis, and CNS damage. Five patients with pyroglutamic aciduria were studied. All presented with hemolytic anemia and metabolic acidosis. Two (brothers) also had Fanconi nephropathy, which is not seen in pyroglutamic aciduria. Molecular analyses of the GSS gene was performed in 3 patients. RT-PCR and heteroduplex analysis identified a homozygous deletion in 1 patient and a homozygous mutation in 2 others (brothers with Fanconi nephropathy). Sequencing of glutathione synthetase (GSS) cDNA from the first patient showed a 141-bp deletion corresponding to the entire exon 4, whilst the corresponding genomic DNA showed a G491 --> A homozygous splice site mutation. Sequencing of GSS cDNA from the Fanconi nephropathy patients showed a C847 --> T [ARG283 --> CYS] mutation in exon 9.

Application of electrospray tandem mass spectrometry to neonatal screening.

For the past 30 years, neonatal screening programs have been performed largely by using the bacterial inhibition assays developed by Dr Robert Guthrie. These programs focused on a small number of diseases such as phenylketonuria and maple syrup urine disease and involved one test for each disease. During the same period many new diseases were discovered, such as organic acidemias and fatty acid oxidation defects, and they presented a diagnostic challenge to biochemical laboratories. Different mass spectrometric approaches have been the main tools for the diagnosis; however, each has its own limitation. Recently, electrospray tandem mass spectrometry (MS/MS) has provided an alternative automated high throughput, specific, and broad-spectrum approach to screening for a relatively large number of disorders, including those covered by bacterial inhibition assays tests. By using specific scan functions, a large number of amino acids and acylcarnitines in blood spots are quantified in 2 minutes analytical time. A new scan function is described here for quantification and screening for argininosuccinic acid in blood spots, which is a key metabolite in the diagnosis of argininosuccinase deficiency. We describe the results of a 3-year tandem MS/MS-based neonatal study that was performed in our newborn population. We screened 27,624 blood spots and identified 20 cases yielding a frequency of 1:1,381. No false-negative cases were identified, but several false-positive cases were eliminated by repeat analysis by MS/MS of blood or by other means. We also used MS/MS analysis of urine or blood either for confirmation of initial positive results or for follow-up of treatment, such as in glutaric acidemia, citrullinemia, argininosuccinase deficiency, and biopterin-dependent phenylketonuria.

Screening blood spots for argininosuccinase deficiency by electrospray tandem mass spectrometry.

Argininosuccinase deficiency is relatively more common in Saudi Arabia than other urea cycle detects (UCD) and its presentation is usually acute and virtually identical to the clinical presentation of other UCD. We developed a rapid, sensitive, and specific screening method for the diagnosis of argininosuccinase deficiency from blood spots. using electrospray tandem mass spectrometry. A 96-well microplate batch process is used for extraction of argininosuccinic acid (ASA), other amino acids and acylcarnitines (Rashed et al. 1995). ASA and other metabolites are derivatized to the corresponding butyl derivatives. The tris-butyl ester of ASA (MH = 459.3) yields two major fragments at m/z 70 and m/z 144 under mild collision induced collision. montitored in the product ion spectrum using a narrow mass range (65-150 kDa). A processing algorithm "CAMPA" is used to automatically calculate the height ratios of selected masses and flags data files as "abnormal" when certain threshold is exceeded. The method is integrated with our existing 2-minute MS/MS method for profiling amino acids and acylcarnitines (Rashed et al. 1997). Using this approach for two years we diagnosed 16 ALD cases from 14 hyperammonemic infants, one high-risk newborn, and one from a regular newborn screening blood spot.

Classic homocystinuria: clinical, biochemical and radiological observations, and therapeutic outcome of 24 Saudi patients.

We considered the clinical, biochemical and radiological findings, and response to pyridoxine (vitamin B6) of 24 classic homocystinuric patients (15 females, 9 males) diagnosed at King Faisal Specialist Hospital and Research Centre. Common clinical findings included ectopia lentis (20 patients), skeletal system involvement (18 patients), vascular system involvement (9 patients) and mental retardation (all patients to varying degrees). A number of unusual findings were reported. The parents of 21 patients were first-degree relatives and 19 patients had at least one other family member affected by the same disease. Only 4 patients responded to pyridoxine; their methionine level decreased to almost normal range.

Tyrosinaemia type II: an easily diagnosed metabolic disorder with a rewarding therapeutic response.

We retrospectively reviewed clinical and biochemical data of four patients diagnosed with tyrosinaemia type II. Diagnosis was established by high plasma tyrosine and normal plasma phenylalanine levels using plasma high-pressure liquid chromatography and tandem mass spectrometry. All patients were mildly mentally retarded and had painful non-pruritic and hyperkeratotic plaques on the soles and palms. There were no ophthalmic symptoms. The patients dramatically responded clinically and biochemically to a diet restricted in tyrosine and phenylalanine.

Tyrosinaemia type II: an easily diagnosed metabolic disorder with a rewarding therapeutic response

We retrospectively reviewed clinical and biochemical data of four patients diagnosed with tyrosinaemia type II. Diagnosis was established by high plasma tyrosine and normal plasma phenylalanine levels using plasma high-pressure liquid chromatography and tandem mass spectrometry. All patients were mildly mentally retarded and had painful non-pruritic and hyperkeratotic plaques on the soles and palms. There were no ophthalmic symptoms. The patients dramatically responded clinically and biochemically to a diet restricted in tyrosine and phenylalanine