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Purpose: Mucopolysaccharidosis IVA (MPS IVA) is a rare lysosomal storage disorder arising from a deficiency in N-acetylgalactosamine-6-sulfatase., Methods: From September 2019 to October 2023, a total of 264,843 Taiwanese newborns underwent screening for MPS IVA using dried blood spots and tandem mass spectrometry., Results: Among the 95 referred infants, 9 (9%) were confirmed to have MPS IVA (group 1), 18 (19%) were highly suspected to have MPS IVA (group 2), 61 (64%) were identified as heterozygotes of MPS IVA (group 3), and 7 (7%) were determined not to have MPS IVA (group 4). A total of 34 different GALNS (HGNC:4122) gene variants were identified through our MPS IVA newborn screening program. The most prevalent variant was c.857C>T p.(Thr286Met), found in 33 cases (29%), followed by c.953T>G p.(Met318Arg) in 22 cases (19%). Intravenous enzyme replacement therapy was initiated in 5 patients at ages ranging from 0.3 to 1.7 years. The estimated incidence of MPS IVA in this screening program was 3.4 per 100,000 live births., Conclusion: Because of the progressive nature of MPS IVA, an early diagnosis facilitated by newborn screening and prompt initiation of enzyme replacement therapy before irreversible organ damage occurs may result in improved clinical outcomes., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Objective: We present detection of hypermethylation at H19 differentially methylated region (DMR) at amniocentesis in a fetus with overgrowth, distended abdomen and Beckwith-Wiedemann syndrome (BWS)., Case Report: A 31-year-old, gravida 2, para 1, woman was referred for genetic counseling at 22 weeks of gestation because of fetal overgrowth with fetal biometry equivalent to 24 weeks of gestation and a distended abdomen with an abdominal circumference equivalent to 26 weeks of gestation. She did not undergo any assisted reproductive technology during this pregnancy. Amniocentesis was performed at 23 weeks of gestation. Conventional cytogenetic analysis revealed a karyotype of 46,XX. Array comparative genomic hybridization analysis on the DNA extracted from uncultured amniocytes revealed no genomic imbalance. Methylation analysis on the DNA extracted from amniocytes revealed hypermethylation at H19DMR [imprinting center 1 (IC1)] and normal methylation at KvDMR1 (IC2). The methylation test confirmed the diagnosis of BWS in the fetus. The parents decided to continue the pregnancy. At 36 weeks of gestation, a 4000-g female baby was delivered with macroglossia, ear tags and creases, and an enlarged liver, consistent with the phenotype of BWS., Conclusion: Prenatal diagnosis of fetal overgrowth should include a differential diagnosis of BWS, and methylation analysis of H19DMR (IC1) and KvDMR1 (IC2) is useful under such a circumstance., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to this article., (Copyright © 2021. Published by Elsevier B.V.)

Despite advances in neonatal intensive care in the recent decade, a large number of very preterm infants (VPIs) remain at risk for significant neurodevelopmental impairment (NDI). Given that there are many interventions need to be implemented during the critical perinatal period so that complications of these vulnerable VPIs could be minimized, it is urgent to develop multi-discipline strategies based on evidence to be carried out. The objective of this new term evidence-based perinatal critical strategies (EBPCS), is to provide beneficial intervention towards better neurodevelopmental outcomes, specifically for preterm infants below 28 weeks gestational age. EBPCS is defined as the management of the VPIs during the perinatal period which would include antenatal counseling with team briefing and share decision making, treat the chorioamnionitis, antenatal MgS04, antenatal steroid, delayed cord clamping/milking, neonatal resuscitation team preparation, prevention of hypothermia, immediate respiratory support with continuous positive airway pressure at delivery room, less invasive surfactant administration, early surfactant with budesonide therapy, support of cardiovascular system, early initiate of probiotics administration, early caffeine, early parenteral and enteral nutrition, promptly initiating antibiotics. These critical strategies will be discussed detail in the text; nonetheless, standardized protocols, technical skills and repeated training are the cornerstones of successful of EBPCS. Further experience from different NICU is needed to prove whether these very complicate and comprehensive perinatal critical strategies could translate into daily practice to mitigate the incidence of NDI in high-risk VPIs., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest relevant to this article., (Copyright © 2020. Published by Elsevier B.V.)

Objective: We present prenatal diagnosis and molecular cytogenetic characterization of an inverted duplication of proximal chromosome 15 [inv dup(15)] presenting as a small supernumerary marker chromosome (sSMC) at amniocentesis associated with concomitant microduplication of 8q22.1., Materials and Methods: A 39-year-old woman underwent amniocentesis at 16 weeks of gestation because of advanced maternal age, and the result was 47, XY, +mar dn. The woman requested for repeat amniocentesis at 20 weeks of gestation. Array comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), quantitative fluorescent polymerase chain reaction (QF-PCR) and DNA methylation analysis were applied to determine the nature of the sSMC., Results: aCGH on the uncultured amniocytes revealed the result of arr 8q22.1 (93,918,763-96,618,539) × 3.0, arr 15q11.2q13.2 (22,765,628-30,658,876) × 4.0, arr 15q13.2q13.3 (30,653,877-32,509,926) × 3.0 [GRCh37 (hg19)]. Interphase FISH analysis using RP11-34H12 [15q13.2; Texas Red, 30,709,033-30,893,021 (hg19)] on 100 uncultured amniocytes showed that 38 cells had three signals, 45 cells had four signals and 27 cells had two signals. The parental bloods had normal aCGH results. The karyotype of cultured amniocytes was 47, XY, +inv dup(15) (pter→q13::q13→pter) which was confirmed by metaphase FISH analysis. No informative markers could be found in QF-PCR analysis. DNA methylation analysis on cord blood confirmed a maternal origin of the 15q11-q13 gene dosage increase with a result of 15q11.2 SNRPN DNA hypermethylation. Postnatal cytogenetic analysis on cord blood, umbilical cord and placenta showed the results consistent with the prenatal diagnosis., Conclusion: Molecular cytogenetic techniques are useful for rapid diagnosis of an inv dup(15) chromosome presenting as an sSMC at amniocentesis., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest relevant to this article., (Copyright © 2020. Published by Elsevier B.V.)

The ductus arteriosus is likely to close without treatment in most infants born at gestational age (GA) > 28 weeks (73%), and those with birth weight > 1000 g (94%). However, the rates of spontaneous ductal closure among less mature or smaller infants with respiratory distress syndrome are not known. Extremely preterm infants born at GA < 28 weeks are associated with a high risk of severe intraventricular hemorrhage (IVH) or pulmonary hemorrhage, which usually occur within 72 h after birth and affect mortality and long-term neurological development. These serious hemorrhagic complications may be closely related to hemodynamic changes caused by a hemodynamically significant patent ductus arteriosus (hs-PDA). While prophylactic indomethacin has been shown to reduce the rates of PDA, PDA ligation, severe IVH and early pulmonary hemorrhage, the available evidence does not support its prophylactic use in preterm infants. Symptomatic or late treatment is associated with lower success rate, and increased complications of a hs-PDA. The issue of "to treat or not to treat a PDA" is controversial. Considering the relationship between the effectiveness and timing of pharmacological treatment, early targeted treatment may be an alternative approach for the early identification of a hs-PDA in specific high-risk patient population, especially infants <26 weeks GA who are at the highest risk of severe IVH or pulmonary hemorrhage. Serial echocardiographic studies can be used to select patients who are candidates for early targeted medical treatment of hs-PDA. Surgical ligation of PDA, and transcatheter closure if proven to be safe, can be used as back-up therapy for patients who fail medical treatment and continue to have cardiopulmonary compromise., (Copyright © 2019. Published by Elsevier B.V.)

Background: Unexplained developmental delay or intellectual disability (DD/ID) has an estimated prevalence of about 3%-5% in the general population of Taiwan. Array comparative genomic hybridization (array-CGH) is a high-resolution tool that can detect about 50 Kb chromosome aberrations. A previous study has reported a detection rate of 10%-20% for this array. 1 This study aimed to investigate and compare the diagnosis rate for DD/ID using array-CGH and conventional chromosome study in DD/ID patients in Taiwan., Methods: We enrolled 177 patients with DD/ID who underwent array-CGH examination at the MacKay Memory Hospital between June 2010 and September 2017. The copy number variants (CNV) were classified into the following three groups: pathogenic (potential pathologic variant), benign (normal genomic variant), and uncertain clinical significance (variance of uncertain significance, VOUS), according to the ACMG guideline. 2 RESULTS: Of the 177 enrolled patients, 100 (56.5%) were men and 77 (43.5%) were women. Ages ranged from 3 months to 50 years, with a median age of 5.2 years. Total 32.0% (32/100) male patients had pathogenic CNV, and 32.5% (25/77) female patients had pathogenic CNV. The ratio of pathogenic CNV in male and female patients was not significantly different (p = 0.379). The proportions of pathogenic CNV at <3 years, 3-6 years, 6-12 years, 12-18 years, and >18 years of age were 32.3% (31/96), 19.4% (6/31), 34.8% (8/23), 16.7% (2/12), and 66.7% (10/15), respectively. The overall diagnosed rate of DD/ID with pathogenic CNV was 27.7% (49/177) using array-CGH in this study. There were 105 patients with conventional karyotyping and array-CGH data at the same time. Nineteen (18.1%) patients had visible chromosomal abnormality. Total 32/105 (30.5%) patients could find at least one pathogenic CNVs. The array-CGH had a higher diagnosed rate than the conventional karyotyping in clinical application., Conclusions: Although array-CGH could not detect point mutation, balanced translocations, inversions, or low-level mosaicism, the diagnosis rate in clinical application was up to 46.3% and 2.5 times that of conventional karyotyping analysis (18.1%). This study demonstrated that array-CGH is a powerful diagnostic tool and should be the first genetic test instead of conventional karyotyping analysis for patients with unexplained DD/ID., (Copyright © 2018. Published by Elsevier B.V.)

Chorioamnionitis is a common cause of preterm birth and may cause adverse neonatal outcomes, including neurodevelopmental sequelae. Chorioamnionitis has been marked to a heterogeneous setting of conditions characterized by infection or inflammation or both, followed by a great variety in clinical practice for mothers and their newborns. Recently, a descriptive term: "intrauterine inflammation or infection or both" abbreviated as "Triple I" has been proposed by a National Institute of Child Health and Human Development expert panel to replace the term chorioamnionitis. It is particularly important to recognize that an isolated maternal fever does not automatically equate to chorioamnionitis. This article will review the current literature on chorioamnionitis, and introduce the concept of Triple I, as well as recommendations for assessment and management of pregnant women and their newborns with a diagnosis of Triple I., (Copyright © 2017. Published by Elsevier B.V.)

Background: Previous studies have identified preterm birth and/or small for gestational age (SGA) as risk factors for features of the metabolic syndrome, including high blood pressure, insulin sensitivity and atherosclerosis, occurring later in life, with controversial results. We conducted this population-based cohort study to investigate metabolic outcomes in those with former preterm birth and/or SGA status in Taiwan., Methods: Data were obtained from Taiwan's universal National Health Insurance Research Database. From 1996 to 2004, 37,119 preterm infants, 3386 SGA infants, and 162,020 matched controls were included. We investigated the risk of the metabolic disease, including hypertension, diabetes, and hyperlipidemia, which had been recorded by the end of 2008., Results: The preterm and SGA cohort, combined into one, had a significantly increased risk of developing metabolic disorders when compared with the comparison cohort (HR = 2.46, 95% CI = 2.02-3.01). We observed that children with former preterm and SGA status in Taiwan had a higher risk of developing hypertension (HR = 3.24, 95% CI = 1.58-6.67), Type 1 diabetes mellitus (HR = 1.80, 95% CI = 1.05-3.07), Type 2 diabetes mellitus (HR = 2.49, 95% CI = 1.98-3.14), and hyperlipidemia (HR = 2.14, 95% CI = 1.29-3.52)., Conclusion: Our study revealed the risk of metabolic disease in those with preterm birth and/or SGA. Further studies with a longer duration of follow-up are required to confirm if there is a tendency for the metabolic syndrome to develop in this study cohort., (Copyright © 2017. Published by Elsevier B.V.)

Menkes disease is a rare neurodegenerative disorder caused by mutations in ATP7A gene. Deficiency in copper-dependent enzymes results in the unique kinky hair appearance, neurodegeneration, developmental delay, seizures, failure to thrive and other connective tissue or organ abnormalities. Other than biochemical tests, DNA-based diagnosis is now playing an important role. More than two hundred mutations in ATP7A gene were identified. Early copper supplementation can help improve neurological symptoms, but not non-neurological problems. Further molecular studies are needed to identify additional mutation types and to understand the mechanism of pathogenesis. This may help in discovering the possible treatment measures to cure the disease. We present a case with the clinical features and biochemical findings, abnormal brain magnetic resonance imaging as well as the effects of treatment with copper-histidine. Direct sequencing of ATP7A gene revealed a de novo point mutation which resulted in an early stop codon with truncated protein., (Copyright © 2014. Published by Elsevier B.V.)

Background: Infantile hypertrophic pyloric stenosis (IHPS) is a common disease in infancy. Pyloromyotomy is universally considered the treatment for IHPS; however, oral or intravenous atropine has been reappraised for the treatment of IHPS in the past 20 years. We investigated the efficacy of atropine in the medical management of IHPS by using meta-analysis and investigated the sonographic changes of the pyloric canal, as well as the efficacy and adverse effects of atropine., Methods: Information was retrieved from PubMed, Ovid, and MEDLINE. The efficacy and adverse effects of atropine treatment for IHPS were reviewed using the standard process of meta-analysis., Results: Eleven articles were obtained. Five reports showed that 77 of 110 (70%) infants who were administered oral atropine benefitted by the induced remission of IHPS. Six reports showed that 288 of 345 (83.5%) patients who were treated initially with intravenous atropine then changed to oral atropine showed beneficial effects and had no serious side effects. Time to pyloric muscle normalization ranged from 5 weeks to 15 months., Conclusion: The study results indicate that atropine is a possible alternative treatment for IHPS, particularly in infants with major concurrent disease, and is safe without obvious side effects., (Copyright © 2016. Published by Elsevier B.V.)

The goal of modern neonatal care of extremely preterm infants is to reduce mortality and long-term neurological impairments. Preterm infants frequently experience cerebral intraventricular or pulmonary hemorrhage, which usually occurs within 72 hours after birth and can lead to long-term neurological sequelae and mortality. These serious hemorrhagic complications are closely related to perinatal hemodynamic changes, including an increase in the afterload on the left ventricle of the heart after the infant is separated from the placenta, and an increased preload from a left-to-right shunt caused by a hemodynamically significant patent ductus arteriosus (PDA). The left ventricle of a preterm myocardium has limited ability to respond to such an increase in afterload and preload, and this can result in cardiac dysfunction and hemodynamic deterioration. We suggest that delayed umbilical cord clamping or umbilical cord milking to maintain optimal blood pressure and systemic blood flow (SBF), careful assessment to keep the afterload at an acceptable level, and a strategy of early targeted treatment of significant PDA to improve perfusion during this critical time period may reduce or prevent these serious complications in preterm infants., (Copyright © 2016. Published by Elsevier B.V.)

Background & Aims: Many studies have reported that serum total homocysteine (tHcy) levels in cystathionine-beta-synthase (CBS) carriers are usually normal and only elevated after a methionine load. However, the amount of methionine required for a loading test is non-physiological and is never reached with regular feeding. Therefore, CBS carriers do not seem to be at an increased risk of cardiovascular diseases. However, the risk of cardiovascular diseases of CBS carriers with folate deficiency has not been studied. We recently found an extraordinarily high carrier rate (1/7.78) of a novel CBS mutation (p.D47E, c.T141A) in an Austronesian Taiwanese Tao tribe who live in a geographic area with folate deficiency. We evaluated if the CBS carriers tend to have higher fasting serum tHcy concentrations than non-carriers in presence of folate deficiency., Methods: The serum tHcy and folate levels before and after folate replacement were measured in 48 adult Tao carriers, 40 age-matched Tao non-carriers and 40 age-matched Han Taiwanese controls., Results: The serum tHcy level of the Tao CBS carriers (17.9 ± 3.8 μmol/l) was significantly higher than in Tao non-carriers (15.7 ± 3.5 μmol/l; p < 0.008) and Taiwanese controls (11.8 ± 2.9 μmol/l; p < 0.001). Furthermore, a high prevalence of folate deficiency in the Tao compared with the Taiwanese controls (4.9 ± 1.8 ng/ml vs. 10.6 ± 5.5 ng/ml; p < 0.001) was also noted. Of note, the difference in tHcy levels between the carriers and non-carriers was eliminated by folate supplementation. (carriers:13.65 ± 2.13 μmol/l; non-carriers:12.39 ± 3.25 μmol/l, p = 0.321)., Conclusions: CBS carriers tend to have a higher tHcy level in the presence of folate deficiency than non-carriers. Although many reports have indicated that CBS carriers are not associated with cardiovascular disease, the risk for CBS carriers with folate deficiency has not been well studied. Owing to a significantly elevated level of fasting tHcy without methionine loading, it is important to evaluate the risk of cardiovascular disease in CBS carriers with folate deficiency., (Copyright © 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Background: Previous studies revealed a high incidence of late-onset Fabry disease mutation, IVS4+919G>A, in Taiwan. However, the natural course is largely unclear and suitable biomarkers for monitoring disease progress are unavailable., Methods and Results: Patients carrying IVS4+919G>A or classical Fabry mutations were enrolled in this study. The subjects ranged from newborn to eighty year old adults. Plasma globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3) were measured by LC-MS/MS in subjects to evaluate the sensitivity of these two biomarkers. All adult males and symptomatic females could be distinguished from healthy controls by an elevated plasma lysoGb3 level. The lysoGb3 level was also related to the left ventricular mass considering gender and age (p<0.01). Moreover, approximately 70% of male and 45% of female newborns already had an elevated plasma lysoGb3 level which increased gradually as the subjects got older (p<0.01)., Conclusions: Plasma lysoGb3 is a more sensitive and reliable biomarker than plasma Gb3. LysoGb3 also correlated with age and left ventricular mass index in Fabry patients with IVS4+919G>A mutation. Because lots of infants with the IVS4+919G>A mutation already had elevated lysoGb3 levels at birth, that indicates that the development of hypertrophic cardiomyopathy may require a long and insidious course after lysoGb3 accumulation., (© 2013. Published by Elsevier B.V. All rights reserved.)

Background: Most premature babies are discharged with low body weight. Creamatocrit represents the lipid concentration of breast milk. We expected the creamatocrit technique could be applied in the nutrition plan for premature infants who were exclusively fed by human milk., Methods: Breast milk samples were obtained from the mothers whose babies were admitted to the neonatal intensive care unit or sick baby room. The breast milk provider was enrolled under the criteria of stable breast milk expression 2 weeks after having given birth. Breast milk was collected for 7 consequent days. Creamatocrit technique and calorie analysis were performed on the processed breast milk samples., Results: Fourteen pairs of mothers and infants were enrolled in our study. The median gestational age and birth weight were 29 weeks (27-36 weeks) and 1,393 g (680-3050 g), respectively. The mean calorie and creamatocrit values for all the 98 breast milk samples were 0.67 kcal/mL and 5.98%, respectively. The linear correlation between creamatocrit value and laboratory-measured calories was found to be calories (kcal/mL)=0.39+0.048×creamatocrit (%) (p<0.05)., Conclusion: We established the relation equation of creamatocrit and calories for the first time in Chinese population, which is convenient and accurate for evaluating calories provided for premature infants fed with breast milk., (Copyright © 2011. Published by Elsevier B.V.)

Lactic acidemia is commonly associated with severe diseases in pediatric patients. Quantitation of blood lactate and pyruvate is important for the diagnosis and clinical management. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using dried blood spots (DBS) was developed and could be used for simultaneous quantification of blood lactate and pyruvate. The applicability of the developed method was tested and confirmed by the regression analysis between LC-MS/MS method and enzymatic assay. Lactate and pyruvate were extracted from DBS obtained from 580 full-term, 120 pre-term infants (gestations ranging from 24 to 36 weeks), and 65 patients with suspected lactic acidemia, with methanolic internal standard (IS) solutions of sodium L-lactate-(13)C(3) and pyruvate-(13)C(3). An API-2000 LC-MS/MS system with multiple reaction monitoring (MRM) mode was applied. The within-run and between-run precisions (CV%) were determined and the results were 1.9% and 3.9% for lactate (n=20) and 5.7% and 7.3% for pyruvate (n=20). The linearity of lactate (r=0.9986) and pyruvate (r=0.9973) based on the IS was excellent. The parameter r squared (r(2)) of linear regression between LC-MS/MS method and enzymatic assay was 0.9405 for lactate and 0.9447 for pyruvate, respectively, and the agreement between these methods was consistent and acceptable. The stability of lactate and pyruvate on DBS was also confirmed. The LC-MS/MS method we developed is a specific, sensitive, and reproducible method for measuring blood lactate and pyruvate concentrations. The use of DBS in this method makes it particularly attractive for pediatric patients.

Background: Information on the long-term efficacy of intravenous pamidronate therapy in Asian patients with osteogenesis imperfecta (OI) is limited. We report our experience using pamidronate in Taiwanese patients with OI., Methods: Twenty-six patients with type I, III, or IV OI (eight males and 18 females; age range at last follow-up, 2.9-39.2 years) who received (or were currently receiving) intravenous pamidronate treatment (30mg/m2/dose, every month) were retrospectively analyzed. Patients were followed for 1.0-7.3 years over the study period from February 2000 to October 2007., Results: The mean standard deviation score (SDS) for bone mineral density (BMD) had increased significantly from -4.72 to -3.37 (p < 0.005) after 1 year of treatment. In 16 patients evaluated after 4 years and eight after 6 years, the mean SDS continued to improve, to -2.69 (p < 0.001) and -1.54 (p < 0.005), respectively. The fracture rate decreased significantly (from 2.8 +/- 1.1 to 0.6 +/- 0.6, p < 0.001), and nine patients (35%) had no fractures while receiving treatment. The response to pamidronate was significantly better in patients with poorer initial BMD SDS (1 year: r = -0.71, p < 0.01; 4 years: r = -0.81, p < 0.01)., Conclusion: This retrospective study suggests that Taiwanese patients with OI can benefit from pamidronate treatment, leading to a reduced incidence of fractures and increased BMD, especially in patients with poor baseline BMD.

Background: Hunter syndrome (mucopolysaccharidosis type II) is an X-linked recessive lysosomal storage disease caused by a defect of the iduronate-2-sulfatase (IDS) gene. The result is impaired IDS enzyme function., Methods: To characterize the biochemical and molecular defects in IDS-deficient patients and their families, we measured IDS enzyme activity by fluorimetric enzyme assay and identified the IDS gene mutations in 14 unrelated Taiwanese patients with varying clinical phenotypes. In addition, haplotype analysis was also performed., Results: Three novel (IVS2+1G>C, 1055del12, and G489D) and 7 previously reported (N63K, P228L, K347E, R468Q, R468W, I485R, and 1241delAG) mutations were found. Together R468Q and R468W account for 42.8% mutations found in our patients. Haplotype analysis using IDS flanking markers DXS1113 and DXS1123 revealed that the unrelated R468Q alleles were independent in origin whereas the unrelated R468W alleles are probably of the same origin. The R468Q mutation in patient 1150 and I485R mutation in patient 710 occurred de novo in male meioses. Once the mutation in a family was identified, restriction analysis was also performed for rapid diagnosis of female carriers in 8 families. Leukocyte IDS measurement revealed significantly wide range of IDS activity in normal controls and MPS II carriers (19.2 - 70.6 vs. 8.4 - 26.6 nmol/h/mg cell protein). The average leukocyte IDS activity of normal controls (n=43) was 43.9+/-13.3 nmol/h/mg protein, whereas patients with MPS II (n=14) had <5% of mean normal IDS activity (0.9+/-0.6 nmol/h/mg protein), and carriers (n=13) had a mean activity of 17.5 (+/-5.7) nmol/h/mg protein. The mean leukocyte IDS activity in female carriers was less than a half of the normal level., Conclusion: Due to a small overlapping range of normal and carriers, the level of enzyme activity cannot be used alone for carrier detection.