same antibody pairs, and each assay is approved ... - Clinical Chemistry

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performance of the automated Access Hybritech PSA and free PSA assays is analytically ... under a variety of sample collection and sample storage conditions. Urology. 1996;48(Suppl ... Paris Cédex 18, France; fax 33-1-40-25-88-21, e-mail.
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Technical Briefs

same antibody pairs, and each assay is approved by the Food and Drug Administration for the same clinical indications. This multicenter evaluation shows that the performance of the automated Access Hybritech PSA and free PSA assays is analytically specific, sensitive, linear, accurate, and precise.

This study was funded by Beckman Coulter, Inc. References 1. McCormack RT, Rittenhouse HG, Finlay JA, Sokoloff RL, Wang TJ, Wolfert RL, et al. Molecular forms of prostate-specific antigen and the human kallikrein gene family: a new era. Urology 1995;45:729 – 44. 2. Van Cangh PJ, De Nayer P, Sauvage P, Tombal B, Elsen M, Lorge F, et al. Free to total prostate-specific antigen (PSA) ratio is superior to total PSA in differentiating benign prostate hypertrophy from prostate cancer. Prostate Suppl 1996;7:30 – 4. 3. Catalona WJ, Partin AW, Slawin KM, Brawer MK, Flanigan RC, Patel A, et al. Use of the percentage of free prostate specific antigen to enhance differentiation of prostate cancer from benign prostatic disease: a prospective multicenter clinical trial. JAMA 1998;279:1542–7. 4. Woodrum DL, Brawer MK, Partin AW, Catalona WJ, Southwick PC. Interpretation of free prostate specific antigen clinical research studies for the detection of prostate cancer. J Urol 1998;159:5–12. 5. National Committee for Clinical Laboratory Standards. Evaluation of precision performance of clinical chemistry devices; approved guideline. NCCLS guideline EP5-A. Wayne, PA: NCCLS, February 1999. 6. National Committee for Clinical Laboratory Standards. Interference testing in clinical chemistry; proposed guideline. NCCLS guideline EP7-P. Villanova, PA: NCCLS, August 1986. 7. Woodrum DL, French C, Shamel LB. Stability of free PSA in serum samples under a variety of sample collection and sample storage conditions. Urology 1996;48(Suppl 6A):33–9. 8. Woodrum DL, York L. Two-year stability of free and total PSA in frozen serum samples. Urology 1998;52:247–51. 9. Blase AB, Sokloff RL, Smith KM. Five PSA methods compared by assaying samples with defined PSA ratios [Technical Brief]. Clin Chem 1997;43: 843– 4. 10. Strobel SA, Sokoloff RL, Wolfert RL, Rittenhouse HG. Multiple forms of prostate specific antigen in serum measured differently in equimolar-and skewed-response assays [Letter]. Clin Chem 1995;41:125–7. 11. Semjonow A, Oberpenning F, Brandt B, Zechel C, Brandau W, Hertle L. Impact of free prostate-specific antigen on discordant measurement results of assay for total prostate-specific antigen. Urology 1996;48:10 –5. 12. Sokoll LJ, Chan DW. Total, free, and complexed PSA: analysis and clinical utility. J Clin Ligand Assay 1998;21:171–9.

Congenital Disorder of Glycosylation Ia with Deficient Phosphomannomutase Activity but Normal Plasma Glycoprotein Pattern, Thierry Dupre,1 Maryvonne Cuer,1 Sandrine Barrot,1 Anne Barnier,1 Vale´rie Cormier-Daire,2 Arnold Munnich,2 Genevie`ve Durand,1,3 and Nathalie Seta1,4* (1 Laboratoire de Biochimie A, Hoˆpital Bichat, 75877 Paris Ce´dex 18, France; 2 Service de Ge´ne´tique Me´dicale, INSERM U393 Hoˆpital Necker, 75015 Paris, France; 3 Faculte´ de Pharmacie, Universite´ Paris XI, 92296 ChaˆtenayMalabry Ce´dex, France; 4 Faculte´ de Pharmacie, Universite´ Paris V, 75006 Paris, France; * address correspondence to this author at: Laboratoire de Biochimie A, Hoˆpital Bichat-Claude Bernard, 46, rue Henri Huchard, 75877 Paris Ce´dex 18, France; fax 33-1-40-25-88-21, e-mail [email protected]) Congenital disorders of glycosylation [CDG; previously carbohydrate-deficient glycoprotein syndrome (1 )] repre-

sent a newly delineated group of inherited diseases (2 ). The CDG are now clearly classified in two groups including subgroups. CDG I, by far the most common type with ⬎300 patients described in the literature, is characterized by defects in the assembly of dolichol pyrophosphate oligosaccharide and/or in the transfer of oligosaccharide from dolichol pyrophosphate to an Asn residue on the nascent proteins. The other group, CDG II, reflects defects in the processing of protein-bound glycans. Only a few cases have been described (1 ). The diagnosis of CDG I is based on biochemical changes involving a unique carbohydrate deficiency observed in serum transferrin (TRF). In healthy subjects, serum TRF is fully glycosylated, containing two N-glycan chains, whereas in CDG I patients, it is partially (one chain) or totally deglycosylated (3 ). This structural abnormality is associated with different enzyme deficiencies (4 ). The most common, subtype Ia, is a deficiency of phosphomannomutase (PMM; EC 5.4.2.8) (5 ) and is present in 70% of CDG I patients. The disease is linked to chromosome 16p13, and numerous missense mutations have been identified in the PMM2 gene (6, 7 ). The condition is an autosomal recessive multisystemic disorder affecting the nervous system and numerous organs, including the liver, kidney, heart, adipose tissue, bone, and genitalia (4 ). The characteristic biochemical abnormalities of CDG can be demonstrated by various methods, including microanion-exchange chromatography or isoelectric focusing of TRF (8 ), based on sialic acid content, and Westernblot analysis of plasma glycoproteins (9 ), based on variations of protein molecular weight. Fig. 1A shows typical isoelectric focusing patterns for serum from a healthy subject and a CDG I patient; Fig. 1B shows typical Western-blot patterns for serum TRF, ␣1-antitrypsin, haptoglobin, and ␣1-acid glycoprotein from a healthy subject and a CDG I patient. The detection limit of the Westernblot method, tested by serial dilution, was ⬍1 ng on the gel regardless of the glycoprotein tested. No discordance was observed between the TRF Western-blot assays and isoelectric focusing when ⬎20 CDG I patient patterns were compared (data not shown). We report here two cases of CDG Ia for which the condition could not be detected as easily as usual. In the first family (F1), the sibling pair was composed of a 16-year-old girl (F1J) and a 6-year-old boy (F1D). Both have classical clinical features of CDG I, including psychomotor retardation, cerebellar ataxia, strabismus, and cerebellar hypoplasia; the girl also has hypogonadism. When Western blotting of the four different glycoproteins was performed on sera from both children, the results were puzzling. The boy’s results showed a characteristic CDG I pattern (Fig. 1B, lane 4), consistent with the clinical features. By contrast, the pattern of serum glycoproteins of the girl (Fig. 1B, lane 3) was identical to the one of healthy subjects. The serum carbohydrate-deficient transferrin (CDT) was measured for both siblings (reference interval, 10 –30 units/L CDT; F1J, 38 units/L CDT; F1D, 148 units/L CDT) and was consistent with the results of

Clinical Chemistry 47, No. 1, 2001

the Western-blot analysis. These results were also confirmed by the isoelectric focusing pattern (Fig. 1A). Three months later, the results were confirmed on new serum samples. During the intervening period, PMM activity was measured according to the method of Van Schaftingen and Jaeken (5 ) on mononucleated leukocytes and on cultured skin fibroblasts from both children. The results obtained from the two cell types demonstrated undetectable PMM activity for both children. Identical mutations

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of the PMM2 gene, R141H and T226S as determined by complete sequencing of cDNA, were found in both children. In the other family (F2), the sibling pair was composed of two adult men (F2L and F2T) with nonprogressive cerebellar ataxia. The Western-blot pattern of the serum glycoproteins from F2T (Fig. 1B, lane 6) was typical for CDG I. In contrast, fewer bands or paler lower bands were found for F2L (Fig. 1B, lane 5). Similarly, the isoelectric

Fig. 1. Isoelectric focusing patterns of serum TRF (A) and Western blots of serum glycoproteins (B) from a healthy subject (lane 1), a CDG Ia patient with a typical electrophoretic pattern (lane 2), and the CDG Ia patients F1J (lane 3), F1D (lane 4), F2L (lane 5), and F2T (lane 6). (B), AAT, ␣1-antitrypsin; HPT, haptoglobin; AGP, ␣1-acid glycoprotein.

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focusing patterns showed a characteristic CDG I profile for F2T but only a partially abnormal one for F2L (Fig. 1A). PMM activity measured in the leukocytes of both patients was undetectable, corresponding to an identical double mutation on the PMM2 gene, R141H and C9Y as determined by complete sequencing of cDNA. Until now, the diagnosis of CDG I has been based on clinical features and confirmed by the presence of abnormally glycosylated serum glycoproteins. Considering our results, we are facing a new situation: patients who have clinical CDG I features and belong to families in which other relatives are clinically and biologically CDG I patients, but who have either intermediate electrophoretic patterns corresponding to glycoproteins lacking fewer glycan chains, or even non-CDG patterns corresponding to normally glycosylated serum glycoproteins. In the first family, the patient F1J with the normal pattern is almost an adult, and the results can be related to those observed in adult patients (10 ). Stibler et al. (10 ) reported that concentrations of CDT are profoundly increased in all patients but tend to be lower in adults than in patients younger than 15 years, with a loss of correlation with age in older patients. The normalization of the glycoprotein glycan content could reflect an adaptation to the metabolic abnormalities. In the case of the 16-year-old patient, the adaptation could be complete although the PMM activity was deficient. In the other family, age does not explain the findings of a typical CDG I pattern in one sibling and, in the other, a pattern with fewer or paler lower bands for all glycoproteins tested, despite similar clinical presentations for both subjects. In conclusion, we have seen at least one clinically confirmed CDG Ia patient with normal serum glycoproteins. The diagnosis of CDG Ia presently based on the evidence of abnormal glycosylation of serum glycoproteins, whatever the method used, might lack sensitivity when applied to teenagers or adults. Biologists who are involved in the diagnosis of CDG should be aware of the possibility of false-negative results. References 1. Aebi M, Helenius A, Schenk B, Barone R, Fiumara A, Berger EG, et al. Carbohydrate-deficient glycoprotein syndromes become congenital disorders of glycosylation: an updated nomenclature for CDG. First International Workshop on CDGS. Glycoconj J 2000;16:669 –71. 2. Jaeken J, Carchon H. The carbohydrate deficient glycoprotein syndromes: an overview. J Inherit Metab Dis 1993;16:813–20. 3. Yamashita K, Ideo K, Ohkura T, Fukushima K, Yuasa I, Ohno K, Takeshita K. Sugar chains of serum transferrin from patients with carbohydrate deficient glycoprotein syndrome. J Biol Chem 1993;268:5783–9. 4. Jaeken J, Matthijs G, Barone R, Carchon H. Carbohydrate deficient glycoprotein (CDG) syndrome type I. J Med Genet 1997;34:73– 6. 5. Van Schaftingen E, Jaeken J. Phosphomannomutase deficiency is a cause of carbohydrate deficient glycoprotein syndrome type I. FEBS Lett 1995;377: 318 –20. 6. Matthijs G, Schollen E, Pardon E, Veiga-Da-Cunha M, Jaeken J, Cassiman J-J, Van Schaftingen E. Mutations in PMM2, a phosphomannomutase gene on chromosome 16p13, in carbohydrate deficient glycoprotein type 1 syndrome [Letter]. Nat Genet 1997;16:88 –92. 7. Vuillaumier-Barrot S, Hetet G, Barnier A, Dupre´ T, Cuer M, de Lonlay P, et al. Identification of four novel PMM2 mutations in congenital disorders of glycosylation (CDG) Ia French patients. J Med Genet 2000;37:579 – 80. 8. Stibler H, Jaeken J, Kristiansson B. Biochemical characteristics and diagno-

sis of the carbohydrate deficient glycoprotein syndrome. Acta Paediatr Scand 1991;375:21–31. 9. Seta N, Barnier A, Hochedez F, Besnard M, Durand G. Diagnostic value of Western blot in carbohydrate-deficient glycoprotein syndrome. Clin Chim Acta 1996;254:131– 40. 10. Stibler H, Blennow G, Kristiansson B, Lindehammer H, Hagberg B. Carbohydrate deficient glycoprotein syndrome: clinical expression in adults with a new metabolic disease. J Neurol Neurosurg Psychiatry 1994;57:552– 6.

A Precaution in the Detection of Heterozygotes by Sequencing: Comparison of Automated DNA Sequencing and PCR-Restriction Fragment Length Polymorphism Methods, Mehmet Simsek,1* Musbah O.M. Tanira,2 Khalid A. Al-Baloushi,2 Hameeda S. Al-Barwani,1 Khulood M. Lawatia,1 and Riad A.L. Bayoumi1 (Departments of 1 Biochemistry and 2 Pharmacology, Sultan Qaboos University, College of Medicine, Muscat, Oman; * address correspondence to this author at: Sultan Qaboos University, College of Medicine, Department of Biochemistry, PO Box 35, Al-Khod, Postal code 123, Muscat Sultanate of Oman; fax 968-513-419, email [email protected]) Single-nucleotide polymorphisms have been detected by various methods, including allele-specific oligonucleotide hybridization (1 ), allele-specific amplification (2 ), and restriction fragment length polymorphism (RFLP) analysis (3 ). In some cases, sequencing of amplified DNA has been used as a direct method (4, 5 ). RFLP-based methods appear to be superior in genotyping studies. In fact, recently, Cascorbi and Roots (6 ) recommended the use of RFLP in preference to other methods for single-nucleotide polymorphism detection in the N-acetyltransferase-2 (NAT2) gene. Some groups have used more than one method in their analyses, and when discrepancies occurred between two methods, they used DNA sequencing as a gold standard (7 ). In this report, we describe a comparison of RFLP methods with automated DNA sequencing for the detection of three different mutations in the NAT2 gene. Our results indicate that caution is needed in the use of automated sequencing to detect heterozygous mutations accurately at some polymorphic sites in the NAT2 gene. Established PCR-RFLP methods were used to detect NAT2 mutations at the C282T, T341C, and C481T sites. The C-to-T mutations at the 282 and 481 sites were detected with RFLP analysis of a 360-bp DNA fragment with FokI or KpnI digestions, respectively (3 ). Two different RFLP methods were used for detection of the T341C mutation: a previously described DdeI method (3 ), and a new complementary NcoI-RFLP. For both methods, a seminested PCR was performed to amplify a 360-bp DNA fragment using a 998-bp DNA fragment as a template. The latter fragment was preamplified as described previously by Hickman and Sim (8 ), using their Nat-Hu14 and NatHu16 primers. The seminested PCR mixture (50 ␮L) contained 2 ␮L of 100-fold diluted 998-bp DNA, 200 ␮M each dNTP, 1.0 ␮M primers, 1.25 U of Taq DNA polymerase (Life Technologies), and a buffer consisting of 10 mM