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Newborn screening for galactosemia: a review of 5 years of data and audit of a revised reporting approach.

Newborn (NB) [4] screening for galactosemia is done primarily to detect clinically devastating galactosemia due to defective function of galactose-1-phosphate uridyltransferase (GALT) (1). Increases in blood galactose (Gal) are also observed in other conditions, however: in the relatively rare galactokinase (GALK) deficiency, which can have serious sequelae; in partial GALT deficiency, which has no clinical consequences; and in UDP-galactose-4-epimerase (GALE) deficiency, which has 1 common benign form and 1 extremely rare untreatable form with severe clinical outcomes (2, 3). Additionally, there are other transient galactosemias of unknown cause and other known benign variants that are routinely flagged in NB screening. Recently, Sakura et al. reported that congenital portal-systemic shunt, rather than hereditary galactosemias, is the most common cause of persistent hypergalactosemia in NB screening in Hiroshima, Japan (4). The Duarte/classic compound heterozygote variant (D/G galactosemia) is often the cause of hypergalactosemia and is frequently flagged as positive in NB-screening programs. The urgent reporting of an increased Gal concentration, with or without GALT deficiency, sets off a chain of events: immediate notification of the Department of Health, primary care physicians, and/or metabolic specialists; bringing the NB in for observation; confirmatory testing; and possible diet change. These events are often nonproductive, wasteful of medical resources, and traumatic for families.

To assess the specificity of galactosemia screening, we compiled NB-screening data in our laboratory from January 1,2001, to March 1,2006, for >1.3 x [10.sup.6] NBs. These data included Gal and GALT measurements for all screened NBs. Samples with an increased Gal concentration and/or low GALT activity were also assayed for galactose 1-phosphate (Gal-1-P) and subjected to qualitative DNA testing for 4 common mutations. Whenever possible, clinical outcomes were obtained. The database summarized here for the initial phase of the study was provided as a starting point for a series of discussions in 2006 that were headed by the Pennsylvania Department of Health (PA DOH) and carried out in conjunction with the laboratory and directors of the 2 Pennsylvania galactosemia treatment centers. The goal of the discussions was to analyze these data within the clinical contexts in order to match the reporting of galactosemia with the clinical phenotype. Combining the patterns provided by mining the large amount of data with the clinical experience of the treatment centers suggested changes in the reporting and subsequent revision of some follow-up protocols. For nonclassic galactosemias, our data suggested that the new protocol would produce fewer urgent referrals, conserve time and effort, reduce financial costs, and reduce emotional stress for a substantial number of families. We implemented the recommended reporting and protocol changes in 2006 and have tracked outcomes for 2 years (2007 and 2008). We report our combined results for both phases of the study.

Materials and Methods

Whatman 903-type filter papers (previously Schleicher & Schuell 903) were used for all collections and all assays.

TOTAL Gal

Total Gal (free Gal plus Gal-1-P) was measured by first extracting a filter paper punch (3/16th in) of a blood spot from each NB. The Gal enzymatic assay uses the Astoria-Pacific automated SPOTCHECK continuous-flow assay system as modified by Hoffman et al. (5). Alkaline phosphatase converted the Gal-1-P in the sample to Gal in the first incubation step. The Gal conversion to galactonolactone by the [NAD.sup.+]-NADH-coupled Gal dehydrogenase reaction was then monitored by the change in NADH fluorescence. The mean total Gal concentration for a representative Pennsylvania NB population is 0.200 mmol/L (3.6 mg/dL) [nonparametric range of 2 SDs, 0.128-0.483 mmol/L (2.3-8.7 mg/dL)].

MODIFIED Gal

Modified Gal was measured in samples with an increased total Gal concentration by repunching the blood spot and reanalysis with the Astoria-Pacific SPOTCHECK system, but without the alkaline phosphatase incubation of the first sample. Subsequent incubation with Gal dehydrogenase yielded NADH fluorescence due to the free Gal in the sample. The Gal-1-P concentration was measured by subtracting the total Gal concentration from the modified Gal concentration. Percentage Gal-1-P was calculated as the Gal-1-P concentration divided by total Gal concentration. The expected Gal-1-P percentage is >25% of the total Gal concentration. If the total Gal concentration is increased, a Gal-1-P value >25% suggests possible GALE deficiency, whereas a Gal-1-P value <25% suggests possible GALK deficiency.

GALT ANALYSIS

GALT activity was measured by first extracting a filter paper punch (3/16th in) of a blood spot from each NB. The assay used was the Astoria-Pacific SPOTCHECK continuous-flow assay system as modified by Sturgeon et al. (6). The conversion of UDP-glucose in a series of enzymatic steps to 6-phosphogluconate was coupled to [NADP.sup.+]-NADPH reduction with fluorescence detection. A linear calibration curve was constructed from known NADPH concentrations (in micromoles per liter). Results were expressed in micromoles per liter of fluorescence measured as a direct indication of enzymatic activity. For in-house QC samples, we obtained CVs of 17% for the low-concentration pool (mean, 40 [micro]mol/L) and 12% for the typical-concentration pool (mean, 277 [micro]mol/L). The reference interval for GALT in the NB population is approximately 150-500 [micro]mol/L. A GALT value [less than or equal to] 32 [micro]mol/L is typical in GALT-deficient galactosemia, whereas GALT activity values of 41-120 [micro]mol/L are common with clinically benign mutations. We use "GALT-deficient" throughout this report to denote the laboratory observation of a low enzymatic activity, irrespective of the mutations detected.

DNA ANALYSIS

Three classic mutations in the GALT (galactose-1phosphate uridylyltransferase) gene (Q188R, K285N, and L195P), the S135L mutation, and the Duarte variant (N314D) were detected as previously described (7). DNA analysis was performed for all GALT-deficient samples (i.e., GALT [less than or equal to] 40 [micro]mol/L).

CRITERIA FOR REPORTING POSITIVES

Before September 2006, the PA DOH defined a positive result for non-GALT-deficient galactosemia as a Gal concentration [greater than or equal to] 1.110 mmol/L ([greater than or equal to] 20 mg/dL). The protocol for reporting positive results included immediate notification of the Newborn Screening Program and notification of the primary care physician with a recommendation for immediate referral of the NB to a galactosemia treatment center. Samples with a Gal concentration < 1.110 mmol/L (<20 mg/dL) but [greater than or equal to] 0.8325 mmol/L ([greater than or equal to] 15 mg/dL) were reported as "inconclusive" on the next working day, along with a request for a repeat collection. The revised reporting protocol was implemented on September 1, 2006. In the revised protocol, only cases with a GALT value [less than or equal to] 40 [micro]mol/L or with a Gal value [greater than or equal to] 1.665 mmol/L ([greater than or equal to] 30 mg/dL) were reported as positive. Cases with a GALT value >40 [micro]mol/L and a Gal value [greater than or equal to] 0.8325 mmol/L ([greater than or equal to] 15 mg/ dL) but < 1.665 mmol/L (<30 mg/dL) were reported as inconclusive, along with a request for repeat collection within 72 h. If the NB was on a Gal-free diet before sample collection, he/she was automatically referred to a treatment center. Otherwise, a repeat collection would be made and analyzed. If the repeat Gal value was [greater than or equal to] 1.110 mmol/L ([greater than or equal to] 20 mg/dL) (or GALT [less than or equal to] 40 [micro]mol/L), the second report stated the results as inconclusive and recommended referral of the NB to the treatment center.

COMPARATIVE AUDIT OF THE NEW REPORTING PROTOCOL

In phase 2 of this study, we reviewed galactosemia-reporting results for all NBs in Pennsylvania during the years 2005, 2007, and 2008. All cases reported as positive or inconclusive were investigated. Occurrences of false positives/negatives were documented, and the impact of the changed protocol was assessed by comparing the 2007 and 2008 results with those for 2005 (before the changes in reporting and protocol). This study was performed with the approval of the PA DOH Institutional Review Board.

Results

In the first phase of this study, we carried out an audit of all positive reports of galactosemias to categorize the laboratory results and then compare them with clinical outcomes. Results were presented to the Pennsylvania Newborn Screening Advisory Committee, and recommendations for changes in reporting were determined. The second phase was a 2-year study (2007, 2008) of outcomes that used the revised reporting and follow-up protocol.

Fig. 1 illustrates the distribution of all of our screen-positive samples for the period January 1,2001, to March 1, 2006, with respect to test results. For the period surveyed, 209 samples had a Gal value [greater than or equal to] 1.110 mmol/L ([greater than or equal to] 20 mg/dL) and/or a GALT value [less than or equal to] 40 [micro]mol/L; 23 of these samples had a GALT activity [less than or equal to] 40 [micro]mol/L. Of the 186 screened samples with a GALT value >40 [micro]mol/L and a Gal concentration [greater than or equal to] 1.110 mmol/L ([greater than or equal to] 20 mg/dL), 176 had a typical Gal-1-P percentage (>25%). Twenty of these 176 cases had a total Gal concentration [greater than or equal to] 1.665 mmol/L ([greater than or equal to] 30 mg/dL), values consistent with possible GALE deficiency, and 10 cases had an abnormally low Gal-1-P percentage ([less than or equal to] 25%). Only 1 of these cases had a Gal value > 1.665 mmol/L (>30 mg/dL), which is consistent with possible GALK deficiency.

[FIGURE 1 OMITTED]

Table 1 summarizes the screening and DNA results for the 23 cases screened between January 1,2001, and March 1, 2006, that had GALT values [less than or equal to] 40 [micro]mol/L. In 14 cases, 2 copies of DNA mutations responsible for GALT deficiency were identified (11 homozygotes, 3 compound heterozygotes). In 4 cases, only 1 DNA mutation was identified (heterozygous G; all individuals with clinical diagnoses of GALT-deficient galactosemia). Case no. 4 was a compound heterozygote with an unidentified exon 10 variant. Case no. 10 was a confirmed case of classic disease that featured no detected mutational markers but did have an apparent partial gene deletion at a location remote from the mutation-probe sites tested. Three cases were D/G heterozygotes, and 1 sample had no detectable mutations (confirmed as classic disease by the symptoms).

Table 2 summarizes the 20 cases in our initial 5-year study that had Gal concentrations [greater than or equal to] 1.665 mmol/L ([greater than or equal to] 30 mg/dL), GALT values >40 [micro]mol/L, and Gal-1-P values >25% (possible GALE deficiency). The cases are grouped by the race of the NB to highlight the observation that the presence of a Duarte mutation in 6 cases was exclusively associated with Caucasian NBs, all with GALT values below the reference interval. For the African American NBs, GALE deficiency was diagnosed in 5 cases. In 2 cases, no remarkable abnormality was noted in the NB period, and in 7 cases (all non-Caucasian), no further follow-up was recorded. Except for the 6 cases with the Duarte mutation, all NBs with Gal concentrations [greater than or equal to] 1.665 mmol/L ([greater than or equal to] 30 mg/dL) and GALT values >40 [micro]mol/L were non-Caucasian.

The analysis of the data in Tables 1 and 2 led to recommendations (see Discussion) for a revised Gal cutoff of 1.665 mmol/L (30 mg/dL) for reporting non-GALT-deficient positives and for revised follow-up protocols for positive and inconclusive screening results (Fig. 2). In the revised protocol, positive screens would require urgent notification and referral to the treatment center, with full dietary restrictions and evaluation/confirmatory testing. Inconclusive screens would not require urgent notification or dietary restrictions until after rescreening, and referral would occur only if the rescreening results were abnormal. The new cutoff and revised protocols were then implemented.

Table 3 compares the galactosemia-screening outcomes obtained in this study's second phase for all NB screens in Pennsylvania for the revised protocol (2007 and 2008) with the screening outcomes obtained with the previous protocol (2005). The total number of NBs increased only slightly, whereas the number of positives requiring urgent calls and referral decreased from 30 cases in 2005 to 5 cases in 2007 and 2 cases in 2008. Inconclusives (next-day calls) remained relatively constant, whereas the number of reported second samples with increased values rose from 10 in 2005 to 18 in 2007 and 20 in 2008, a result that reflects the higher number of cases initially called as inconclusive that continued to have increased Gal values.

Discussion

The screening data for galactosemia presented in Fig. 1 reveal that only 13% of all positive cases reported during the period 2001-2006 were associated with a GALT value [less than or equal to] 40 [micro]mol/L. A review of all cases with GALT values [less than or equal to] 40 [micro]mol/L (Table 1) provides the following observations: We found no GALT-deficient galactosemia when the GALT value was >38 [micro]mol/L. On the basis of either DNA or clinical confirmation, we found 19 cases of GALT-deficient galactosemia, a frequency of approximately 1 in 68 400. The sensitivity for detecting GALT deficiency was 100% with the GALT cutoff of [less than or equal to] 40 [micro]mol/L; the positive predictive value with this cutoff was 83% (19 of 23 cases). No GALT-deficient cases were missed.

[FIGURE 2 OMITTED]

Three cases reported as positive with GALT values [less than or equal to] 40 [micro]mol/L were heterozygous D/G. The vast majority of NBs who are D/G heterozygotes have GALT values in the interval of 41-150 [micro]mol/L (data not shown) and typically show improvement in GALT activity in the weeks after initial screening, as with the NB in case no. 9 in Table 1. GALT DNA analysis is ancillary to screening and potentially a diagnostic aid. When mutations are found, such analysis is most useful for differentiating clinically true GALT deficiency from D/G galactosemia for the purpose of treatment. Treatment of D/G galactosemia is controversial; however, Ficicioglu et al. recently reported that long-term clinical and developmental outcomes in children with D/G galactosemia are good, regardless of any diet changes in the first year of life, and that there was no relationship between clinical/developmental outcomes and the concentrations of erythrocyte Gal-1-P and urine galactitol (8). The GALT cutoff of 40 [micro]mol/L is sufficiently low to exclude most compound heterozygote D/G cases without missing true GALT-deficient galactosemias.

Of the 19 confirmed GALT-deficient galactosemias, we identified 2 GALT mutations in 14 cases (11 homozygotes, 3 compound heterozygotes), and 1 mutation in 4 cases. In 1 case, none of the mutations we tested for were present. With the 4 mutations tested, we expect to confirm 70%-80% of the mutations responsible for GALT deficiency with DNA analysis.

The vast majority (87%) of screen positives for galactosemia in the 5-year study were not GALT deficient. GALT-deficient galactosemias are expected to represent 95% of all true clinically important cases; that is, the frequencies of GALK deficiencies and clinically important GALE deficiencies are small by comparison. Therefore, few of these 186 cases are likely to represent clinically important cases of either of these deficiencies. By determining the Gal-1-P percentage in samples with Gal concentrations [greater than or equal to] 1.100 mmol/L ([greater than or equal to] 20 mg/dL), we were able to segregate these 186 cases into those with possible GALK deficiency (Gal-1-P <25%) and those with a possible GALE deficiency (Gal-1-P [greater than or equal to] 25%). We identified 10 cases of possible GALK deficiencies, of which only 1 case [GALT, 299 [micro]mol/L; total Gal, 3.996 mmol/L (72 mg/dL)] was a confirmed positive. Early differentiation of possible GALK-deficient NBs from possible GALE-deficient NBs with the Gal-1-P percentage is an important additional step in guiding clinical decision-making in these cases.

The remaining 176 cases had results consistent with GALE deficiency. To investigate the possible GALE deficiencies, we carried out a follow-up of the subset of the 176 cases that had total Gal values [greater than or equal to] 1665 mmol/L ([greater than or equal to] 30 mg/dL) (n = 20; see Table 2). The cases are grouped by race to highlight that all of the cases with a Duarte mutation were Caucasian NBs and that all of the remaining cases were non-Caucasian NBs. As expected, NBs who are Duarte heterozygotes or compound heterozygotes often have GALT activities less than the lower limit of 2 SDs (150 [micro]mol/L). All of the confirmed GALE deficiencies (including 1 profound deficiency) were African American NBs. Benign GALE deficiency has been reported to occur with a frequency of approximately 1 in 6200 African Americans and 1 in 64 000 Caucasians (9). On the basis of these numbers, benign GALE deficiency should occur with a frequency comparable to that of classic galactosemia in our population mix. Our identification of only 6 cases may be partially attributed to an inability to obtain final diagnoses for some of the NBs listed in Table 2. This result also suggests that some NBs with benign GALE deficiencies have Gal values < 1.665 mmol/L (<30 mg/dL).

In March 2006, the second phase of the study was formulated. We presented our data in a conference call with representatives from the laboratory, the PA DOH Newborn Screening Program, and galactosemia treatment centers. We reviewed the numbers of cases and their apparent clinical classifications and requested input from the treatment center directors in assessing the appropriateness of the classifications, in defining the degree of urgency of notification and treatment for each category from their perspective, and in providing us with input on the treatment protocols.

The clinical input regarding urgency of reporting and treatment was central to our final recommendations. The focus of the discussion was primarily on the 156 cases among the group with possible GALE deficiency that had been called as positive and referred to galactosemia treatment centers. The treatment center directors commented that in their experience true GALK and GALE deficiencies are associated with very high Gal values, usually >3.330 mmol/L (>60 mg/dL). They proposed that a screening cutoff of 1.665 mmol/L (30 mg/dL) was a reasonably conservative value for detecting clinically important GALE or GALK deficiency. They further proposed that all NBs with Gal values < 1.665 mmol/L (<30 mg/dL) and GALT values >40 [micro]mol/L be considered as nonurgent cases from a notification/treatment perspective. A repeat collection would be sufficient follow-up in these cases, with a persistent abnormal Gal result in the second sample being the signal for referral to a treatment center. One caveat was that any NB with a Gal value [greater than or equal to] 0.8325 mmol/L ([greater than or equal to] 15 mg/dL) who had been on Gal-free formula before sample collection would be automatically referred to a treatment center.

With 1.665 mmol/L (30 mg/dL) as a Gal cutoff, 165 NBs in our first-phase study (156 possible GALE cases plus 9 possible GALK-deficiency cases) with Gal values < 1.665 mmol/L (<30 mg/dL) could have been reported as inconclusive (unless the NB was on a Gal-free diet before collection). Urgent calls with urgent referral to a galactosemia treatment center could have been avoided in these cases.

We developed a flow diagram that reflects the suggested changes (Fig. 2). This flow diagram consolidates all samples with GALT values [less than or equal to] 40 [micro]mol/L and Gal values [greater than or equal to] 1.665 mmol/L ([greater than or equal to] 30 mg/dL) under positive reporting and consolidates all samples with GALT values >40 [micro]mol/L and Gal values [greater than or equal to] 0.8325 mmol/L ([greater than or equal to] 15 mg/dL) but <1.665 mmol/L (<30 mg/dL) as inconclusive. These suggested changes in definitions for positive and inconclusive classification were matched with the proposed revisions in clinical protocols for each classification: A positive screening result would be defined as a GALT-deficient galactosemia or as a Gal value [greater than or equal to] 1.665 mmol/L ([greater than or equal to] 30 mg/dL) and would require urgent notification, referral to a treatment center, full dietary restriction, and appropriate confirmatory testing. An inconclusive screening result would be defined as an initial GALT value >40 [micro]mol/L and a Gal concentration [greater than or equal to] 832.5 mmol/L (15 mg/dL) but <1.665 mmol/L (30 mg/dL) and would not require urgent notification or dietary restriction, but it would require a repeat collection. Referral would then occur only if the NB was on a Gal-free diet before the first collection or if the result of the repeat screen was abnormal. Also included in Fig. 2 is the decision point for the NB-screening counselors to contact the primary care provider in the inconclusive arm to determine if the NB had been on a Gal-free diet before collection of the blood spot. If so, the NB would then be referred immediately to a treatment center.

Table 3 demonstrates that the number of positives (urgent calls) in Pennsylvania decreased substantially from 2005 to 2007 and 2008. Of the 7 reported positives in 2007 and 2008, 3 were GALT-deficient galactosemias, 2 were GALE deficiencies, 1 was a D/G compound heterozygote variant, and 1 was lost to follow-up. For 2008, one of the 2 reported positives was GALT deficient. The other was identified as having 1 copy of a GALT mutation, but this NB was lost to follow-up. An audit of positive urgent calls in 2005 showed that of the 30 reported positives, 5 were true positives (2 GALT cases, 2 GALE cases, and 1 GALK-deficiency case), 7 cases were D/G galactosemia, 12 were ultimately diagnosed as galactosemia negative, 4 were carriers, and 2 were lost to follow-up. Clearly, unnecessary urgent calls on NBs with increased Gal values were eliminated by the protocol change.

The number of NBs with increased Gal values in the second sample (delayed referrals) nearly doubled from 2005 to 2007 and 2008 because of the classification of NBs as inconclusive who would have previously been reported as positive [i.e., Gal values [greater than or equal to] 1.110 mmol/L ([greater than or equal to] 20 mg/dL) and GALT values >40 [micro]mol/L]. Nevertheless, the total number of referrals has been substantially reduced because of the normalization of Gal results with repeat evaluation in many cases.

Eighteen cases in 2007 and 22 cases in 2008 were initially called as inconclusive but had abnormal results in repeat screens. Such cases are delayed referrals under the revised protocol. A review of the final diagnoses in the 2007 cases revealed 8 classic variants (3 D/G heterozygotes, 3 single-copy heterozygotes, 2 Duarte carriers), 1 GALE deficiency, 1 GALE carrier, and 8 cases negative for galactosemia. In 2008, there were 10 variants (5 D/G compound heterozygotes, 3 single-copy heterozygotes, 1 Duarte homozygote, 1 unidentified), 9 cases negative for galactosemia, and 2 cases with a final diagnosis pending. Thus, the repeat screening allowed appropriate identification and referral of cases with persistent Gal increases.

In summary, urgent referrals with the revised protocol during the years 2007 and 2008 were reduced to 5 and 2 cases, respectively, compared with 30 cases in 2005. This dramatic reduction was entirely due to reclassification of urgent reporting for nonclassic galactosemia cases with GALT values >40 [micro]mol/L and Gal values [greater than or equal to] 1.110 mmol/L ([greater than or equal to] 20 mg/dL) but <1.665 mmol/L (<30 mg/dL) from positive to inconclusive. Total referrals were reduced from 41 in 2005 to 23 in 2007 and 24 in 2008. This reduction was due to normalization of Gal results in repeat testing of the reclassified cases. The reduction in referrals represents a substantial savings in terms of the emotional stress on parents, the NB being spared from unnecessary referral, and the costs to the healthcare system.

Conclusion

The primary goal of NB screening for galactosemia is to identify GALT-deficient galactosemia. Semiquantitative GALT analysis with an appropriate cutoff identifies these cases with few false positives. When NB screening includes Gal quantification, the vast majority of reported galactosemias in NBs are nonclassic, however, and these NBs rarely require urgent treatment. The traditional Gal cutoff for galactosemia [1.110 mmol/L (20 mg/dL)] is not optimal for urgent follow-up if the GALT value is >40 [micro]mol/L and leads to unnecessary urgent notification and initiation of treatment protocols, which can be avoided with selective monitoring of second samples. By combining the accumulated data for comprehensive galactosemia testing with the clinical expertise of metabolic specialists in the treatment centers, the PA DOH Newborn Screening Program was able to review and improve follow-up protocols for nonclassic galactosemia, thereby saving money, time, and stress on an already overburdened system.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: D.E. Freer, PerkinElmer Genetics.

Consultant or Advisory Role: D.E. Freer, PerkinElmer Genetics.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: None declared.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We thank Bethany Sgroi-Gaita, Stacy Lloyd, and Meredith Patik, genetic counselors for PerkinElmer Genetics, who investigated tirelessly to obtain clinical data crucial to this work. We also thank Lin Wolf, CRNP, and Nicole Waters, nutritionist at the University of Pittsburgh NB nursery, Irma Payan, CRNP, and NB screening coordinator Daria Tolentino from the University of Pennsylvania NB nursery for their assistance in providing patient information. Finally, we thank Melita Jordan of the PA DOH, who recognized the value of this work and facilitated the discussions.

References

(1.) Cuthbert C, Klapper H, Elsas L. Diagnosis of inherited disorders of galactose metabolism. Curr Protoc Hum Genet 2008;56:17.5.1-29.

(2.) Bosch AM, Bakker HD, van Gennip AH, van Kempen JV, Wanders RJ, Wijburg FA. Clinical features of galactokinase deficiency: a review of the literature. J Inherit Metab Dis 2002;25:629-34.

(3.) Openo KK, Schulz JM, Vargas CA, Orton CS, Epstein MP, Schnur RE, et al. Epimerase-deficiency galactosemia is not a binary condition. Am J Hum Genet 2006;78:89-102.

(4.) Sakura N, Mizoguchi N, Ono H, Nishimura Y, Naito K. Congenital porto-system shunt as the major cause of galactosemia. Int Pediatr 2001;16:206-10.

(5.) Hoffman GL, Laessig RH, Hassemer DJ, Makowski ER. Dual-channel continuous-flow system for determination of phenylalanine and galactose: application to newborn screening. Clin Chem 1984;30: 287-90.

(6.) Sturgeon P, Beutler E, McQuiston D. Automated method for screening galactosemia. In: Technicon Symposia. Automation in analytical chemistry. Vol. 1. White Plains (NY): Mediad; 1966. p 75-7.

(7.) Dobrowolski SF, Banas RA, Suzow JG, Berkley M, Naylor EW. Analysis of common mutations in the galactose-1-phosphate uridyl transferase gene: new assays to increase the sensitivity and specificity of newborn screening for galactosemia. J Mol Diagn 2003;5:42-7.

(8.) Ficicioglu C, Thomas N, Yager C, Gallagher PR, Hussa C, MattieA, etal.Duarte (DG)galactosemia: a pilot study of biochemical and neurodevelopmental assessment in children detected by newborn screening. Mol Genet Metab 2008;95:206-12.

(9.) Alano A, Almashanu S, Chinsky JM, Costeas P, Blitzer MG, Wulfsberg EA, Cowan TM. Molecular characterization of a unique patient with epimerase-deficiency galactosaemia, J Inherit Metab Dis 1998;21:341-50.

Dennis E. Freer, [1] * ([dagger]) Can Ficicioglu, [2] ([dagger]) and David Finegold [3]

[1] Biochemistry Department, PerkinElmer Genetics, Bridgeville, PA; [2] Children's Hospital of Philadelphia, Section of Biochemical Genetics, University of Pennsylvania, Philadelphia, PA; [3] Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA.

* Address correspondence to this author at: PerkinElmer Genetics, Inc., 90 Emerson Lane, Bridgeville, PA 15017. E-mail Dennis.Freer@perkinelmer.com.

([dagger]) These authors contributed equally to this work.

Received September 9, 2009; accepted December 18, 2009.

Previously published online at DOI: 10.1373/clinchem.2009.135947

[4] Nonstandard abbreviations: NB, newborn; GALT, galactose-1-phosphate uridyltransferase; Gal, galactose; GALK, galactokinase; GALE, galactose-4-epimerase; D/G galactosemia, Duarte/classic galactosemia; Gal-1-P, galactose 1-phosphate; PA DOH, Pennsylvania Department of Health.
Table 1. NB-screening cases with GALT values < 40 [micro]mol-L in a 5-
year review.

Case Gal, GALT, DNA (b) Notes
no. mmol/L (a) [micro]mol/L

1 2.80 16 Cpd het
2 1.26 17 Homo G
3 4.21 17.7 Het G Classic
4 3.74 19.9 G/exon 10 variant
5 3.60 20 Homo G
6 1.54 20.2 Homo G
7 3.87 20.5 Het G Classic
8 3.56 21.2 Homo G
9 1.66 22 Het D/G Second GALT, 46
 [micro]mol/L
10 7.19 22.2 WT Second GALT, 12
 [micro]mol/L;
 classic
 (deletion)
11 1.75 22.3 Homo G
12 4.68 22.3 Homo G
13 3.86 25.4 Homo G
14 1.13 26.3 Homo G
15 3.90 27 Homo G
16 3.17 28 Homo G
17 3.26 28.2 Het G Second GALT, 28
 [micro]mol/L;
 classic
18 4.44 29 Homo G
19 1.49 29.3 Het D/G
20 1.64 30 Cpd het
21 9.55 31 Het G Classic
22 3.71 31.3 Cpd het
23 1.30 38.3 Het D/G

(a) To convert Gal concentrations to milligrams per deciliter, divide
by 0.0555.

(b) Cpd het, 2 different GALT mutations (compound heterozygote); Homo
G, 2 GALT mutations (homozygous); Het G, 1 copy of GALT mutation,
other allele unidentified; Het D/G, Duarte/GALT mutation
heterozygotes; WT, wild type (no mutations detected).

Table 2. NBs with values > 1.665 mmol/L (> 30.0 mg/dL) and GALT values
>40 [micro]mmol/L.

Case no. Gal Gal, GALT, Gal-1-P Race
 mmol/L (a) [mmicro]mol/L

1 1.665 315 N (b) A
2 1.70 172 N A
3 1.665 201 N B
4 1.73 175 N B
5 1.73 218 N B
6 1.74 281 N B
7 1.77 182 N B
8 1.78 191 N B
9 1.85 205 N B
10 1.91 191 N B
11 2.01 289 N B
12 2.45 162 N B
13 1.69 63 N C
14 1.71 145 N C
15 1.75 46 N C
16 1.77 82 N C
17 1.85 41 N C
18 1.85 76 N C
19 1.75 277 N O
20 1.80 118 N O

Case no. Clinical

1 No clinical abnormality
2 FNA
3 FNA
4 FNA
5 Epimerase deficiency
6 Epimerase deficiency
7 Epimerase deficiency (mild)
8 FNA
9 FNA
10 FNA
11 Epimerase deficiency (profound)
12 Epimerase deficiency (transient)
13 Het D/G
14 Het D
15 Het D/G
16 Het D
17 Het D/G
18 Het D
19 No abnormality
20 FNA

(a) To convert Gal concentrations to milligrams per deciliter, divide
by 0.0555.

(b) N, within reference interval; A, Asian; FNA, further follow-up
data not available; B, African American; C, Caucasian; Het D/G,
Duarte/GALT mutation heterozygote; Het D, Duarte mutation only
detected; O, other.

Table 3. Outcomes comparison: new protocol vs
previous protocol.

 Year

 2005 2007 2008

Total NBs, n 126 146 136 764 138 196
Positives (urgent calls), n 30 5 2
Inconclusive (next-day call), n (a) 93 95 111
Increased Gal, second samples 11 18 22
 (delayed referral), n
Total referrals, n 41 23 24

(a) GALT > 40 [micro]mol/L; 0.8325 mmol/L (15 mg/dL) [less than or
equal to] Gal < 1.665 mmol/L (30 mg/dL).
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Title Annotation:Pediatric Clinical Chemistry
Author:Freer, Dennis E.; Ficicioglu, Can; Finegold, David
Publication:Clinical Chemistry
Date:Mar 1, 2010
Words:5438
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