Biochemical characteristics of newborns with carnitine transporter defect identified by newborn screening in California.

Carnitine transporter defect (CTD; also known as systemic primary carnitine deficiency; MIM 212140) is due to mutations in the SLC22A5 gene and leads to extremely low carnitine levels in blood and tissues. Affected individuals may develop early onset cardiomyopathy, weakness, or encephalopathy, which may be serious or even fatal. The disorder can be suggested by newborn screening. However, markedly low newborn carnitine levels can also be caused by conditions unrelated to CTD, such as the low carnitine levels often associated with normal pregnancies and some metabolic disorders occurring in the mother. In order to clarify the biochemical characteristics most useful for identification of CTD in newborns, we examined California Department of Public Health newborn screening data for CTD from 2005 to 12 and performed detailed chart reviews at six metabolic centers in California. The reviews covered 14 cases of newborn CTD, 14 cases of maternal disorders (CTD, 6 cases; glutaric aciduria, type 1, 5; medium-chain acyl CoA dehydrogenase deficiency, 2; and cobalamin C deficiency, 1), and 154 false-positive cases identified by newborn screening. Our results show that newborns with CTD identified by NBS exhibit different biochemical characteristics, compared to individuals ascertained clinically. Newborns with CTD may have NBS dried blood spot free carnitine near the lower cutoff and confirmatory plasma total and free carnitine levels near the normal lower limit, particularly if obtained within two weeks after birth. These findings raise the concern that true cases of CTD may exist that could have been missed by newborn screening. CTD should be considered as a possible diagnosis in cases with suggestive clinical features, even if CTD was thought to be excluded in the newborn period. Maternal plasma total carnitine and newborn urine total carnitine values are the most important predictors of true CTD in newborns. However, biochemical testing alone does not yield a discriminant rule to distinguish true CTD from low carnitine in newborns due to other causes. Because of this biochemical variability and overlap, molecular genetic testing is imperative to confirm CTD in newborns. Additionally, functional testing of fibroblast carnitine uptake remains necessary for cases in which other confirmatory testing is inconclusive. Even with utilization of all available diagnostic testing methods, confirmation of CTD ascertained by NBS remains lengthy and challenging. Incorporation of molecular analysis as a second tier step in NBS for CTD may be beneficial and should be investigated.

Krabbe disease: clinical, biochemical and molecular information on six new patients and successful retrospective diagnosis using stored newborn screening cards.

PURPOSE: To present clinical, biochemical and molecular information on six new clinically diagnosed Krabbe disease patients and assess the sensitivity of retrospective galactocerebrosidase measurement in their newborn screening samples. METHODS: Medical records were reviewed. Galactocerebrosidase activity was measured in leukocytes and, retrospectively, in the patients' newborn screening cards (stored for 1.4 to 13.5 years). GALC gene mutation analysis was performed. RESULTS: Five patients with Krabbe disease, one of whom also had hydrocephalus, became symptomatic during infancy. A sixth patient presented with seizures and developmental regression at age two and had a protracted disease course. Galactocerebrosidase activity in leukocytes ranged from 0.00 to 0.20 nmol/h/mg protein. Low galactocerebrosidase activity (range: 3.2% to 11.1% of the daily mean), consistent with Krabbe disease, was detected in each of the newborn screening samples. GALC molecular analysis identified six previously unreported mutations and two novel sequence variants. CONCLUSION: Our cases highlight the clinical variability of Krabbe disease. Galactocerebrosidase activity in newborn dried blood spots is a highly sensitive test, even when samples have been stored for many years. The high frequency of private mutations in the GALC gene may limit the use of genetic information for making treatment decisions in the newborn period.

Maternal medium-chain acyl-CoA dehydrogenase deficiency identified by newborn screening.

Prior to the advent of expanded newborn screening, sudden and unexplained death was often the first and only symptom of medium-chain acyl-CoA dehydrogenase deficiency (MCADD). With the use of tandem mass spectrometry, infants can now be identified and treated before a life threatening metabolic decompensation occurs. Newborn screening has also been shown to detect previously undiagnosed maternal inborn errors of metabolism. We have now diagnosed two women with MCADD following the identification of low free carnitine in their newborns. While one of the women reported prior symptoms of fasting intolerance, neither had a history of metabolic decompensation or other symptoms consistent with a fatty acid oxidation disorder. These cases illustrate the importance of including urine organic acid analysis and an acylcarnitine profile as part of the confirmatory testing algorithm for mothers when low free carnitine is identified in their infants.

Maple syrup urine disease: further evidence that newborn screening may fail to identify variant forms.

Newborn screening (NBS) by tandem mass spectrometry (MS/MS) has allowed for early detection and initiation of treatment in many patients with maple syrup urine disease (MSUD) (OMIM 248600), however, a recent report suggests that variants forms may be missed. Information on these patients is limited. We present clinical, biochemical and molecular information on patients with variant forms of MSUD not detected by the California Newborn Screening Program. Between July 2005 and July 2009, 2200,000 newborns were screened in California by MS/MS. Seventeen cases of MSUD were detected and three (two siblings) were missed. Additionally, the NBS cards of two siblings with late onset MSUD, who were born pre-expanded NBS, were retrospectively analyzed. None of the five patients met criteria to be considered presumptive positive for MSUD (leucine>200micromol/L and a ratio of leucine/alanine>or=1.5). Alloisoleucine (allo-ile) was subsequently analyzed in the NBS cards of all five patients, two of whom were found to have elevated levels. The proband in each family was diagnosed following symptoms triggered by an intercurrent illness or increased protein intake. At diagnosis, leucine levels ranged between 561 and >4528micromol/L, and allo-ile ranged from 137 to 239micromol/L. Two affected siblings had normal plasma amino acids when asymptomatic; however, their biochemical profiles were diagnostic of MSUD during intercurrent illnesses. The median age at diagnosis of all patients was one year (range 0.8-6.7). Heterozygous BCKDHB (E1beta) mutations (c.832G>A/c.970C>T) were identified in one family and a homozygous DBT (E2) sequence variant (c.1430 T>G) in another. The third family had one identifiable DBT mutation (c.827T>G), however, a second mutation was not detected. This report provides further evidence that NBS by MS/MS is unable to detect all cases of MSUD. Second-tier testing with allo-ile may improve sensitivity; however, some children with variant forms will invariably be missed.

Adenovirus detection in Guthrie cards from paediatric leukaemia cases and controls.

Archived neonatal blood cards (Guthrie cards) from children who later contracted leukaemia and matched normal controls were assayed for adenovirus (AdV) C DNA content using two highly sensitive methods. In contrast to a previous report, AdV DNA was not detected at a higher frequency among neonates who later developed leukaemia, when compared with controls.

Universal screening for hemoglobinopathies using high-performance liquid chromatography: clinical results of 2.2 million screens.

In screening for hemoglobinopathies, high-performance liquid chromatography (HPLC) achieves excellent sensitivity and specificity, while adding the very important quantitative element to the analysis. Due to the development of a rapid, automated HPLC system, California began screening 600,000 births per year in 1990 using this method. Based on confirmatory testing for 97% of the initial positive results resulting from 2.2 million screens, HPLC has proven to be clinically accurate. In conjunction with the availability of quantitative data, HPLC provides a complete screening system, eliminating the need for a second screening test, and accurately discriminating beta thalassemia compound conditions. The large degree of ethnic diversity and high birth rate in California have produced detailed and reliable birth prevalence rates for most conditions and ethnicities.

Sex ratios in fetuses and liveborn infants with autosomal aneuploidy.

Ten data sources were used substantially to increase the available data for estimating fetal and livebirth sex ratios for Patau (trisomy 13), Edwards (trisomy 18), and Down (trisomy 21) syndromes and controls. The fetal sex ratio estimate was 0.88 (N = 584) for trisomy 13, 0.90 (N = 1702) for trisomy 18, and 1.16 (N = 3154) for trisomy 21. All were significantly different from prenatal controls (1.07). The estimated ratios in prenatal controls were 1.28 (N = 1409) for CVSs and 1.06 (N = 49427) for amniocenteses, indicating a clear differential selection against males, mostly during the first half of fetal development. By contrast, there were no sex ratio differences for any of the trisomies when comparing gestational ages < 16 and > 16 weeks. The livebirth sex ratio estimate was 0.90 (N = 293) for trisomy 13, 0.63 (N = 497) for trisomy 18, and 1.15 (N = 6424) for trisomy 21, the latter two being statistically different than controls (1.05) (N = 3660707). These ratios for trisomies 13 and 18 were also statistically different than the ratio for trisomy 21. Only in trisomy 18 did the sex ratios in fetuses and livebirths differ, indicating a prenatal selection against males > 16 weeks. No effects of maternal age or race were found on these estimates for any of the fetal or livebirth trisomies. Sex ratios for translocations and mosaics were also estimated for these aneuploids. Compared to previous estimates, these results are less extreme, most likely because of larger sample sizes and less sample bias. They support the hypothesis that these trisomy sex ratios are skewed at conception, or become so during embryonic development through differential intrauterine selection. The estimate for Down syndrome livebirths is also consistent with the hypothesis that its higher sex ratio is associated with paternal nondisjunction.

Use of the phenylalanine:tyrosine ratio to test newborns for phenylketonuria in a large public health screening programme.

OBJECTIVE: To assess the benefits of using the phenylalanine:tyrosine ratio to screen newborns for phenylketonuria (PKU). SETTING: Data were collected from all newborns in California during a ten month period (n = 404,381). METHODS: Dried blood spot specimens were analysed at nine laboratories. To assure that the results reported from multiple sites were matched accurately, an automated methodology was chosen that included sample processing, analysis, telecommunications, reporting, and information technology. Phenylalanine and tyrosine concentrations were measured independently by continuous flow fluorometry, for which precision, recovery, detection limits, carryover, chemical specificity, reportable range, and number of repeats are reported. RESULTS: In this study, 37% of the newborns were tested at less than 24 hours of age. For this population, using a phenylalanine only cut off of 200 mumol/l, there were 48 recalled infants per case of classic PKU. Using the phenylalanine:tyrosine ratio with a cut off of 1.50, screen positives could be reported with phenylalanine as low as 150 mumol/l and with only 12 recalls per case. CONCLUSIONS: The phenylalanine:tyrosine ratio can be measured accurately at multiple laboratories using two channel chemical analyses. Having applied the methods to the routine clinical screening of a large population, it was confirmed that the clinical sensitivity and specificity of the PKU screening test are higher when the phenylalanine:tyrosine ratio is incorporated into the cut off than when the cut off is based on the phenylalanine concentration alone.

Neural tube defect prevalence in California (1990-1994): eliciting patterns by type of defect and maternal race/ethnicity.

This study presents race/ethnicity-specific prevalence estimates of neural tube defects (NTDs) in California using 5 years of population-based data. NTD prevalence estimates include prenatally diagnosed cases, as well as cases diagnosed at birth. The California NTD Registry contains NTD case reports identified through the California Maternal Serum Alpha-Feto Protein (AFP) Screening Program, the California Birth Defects Monitoring Program, and additional reports from clinicians and clinics throughout the state. These data were used to estimate NTD prevalence in a large population-based study (n = 1,618,279). The overall NTD prevalence among White, Black, Hispanic, and Asian women are reported, as well as race/ethnic prevalence, for anencephaly, spina bifida, and encephalocele. Rates are expressed as the number of cases per 1,000 women screened between 1990 and 1994. Among 1,457 women with an NTD-affected pregnancy, the overall rate for anencephaly, spina bifida, and encephalocele was 0.49 (95% CI 0.46-0.53), 0.42 (95% CI 0.38-0.45), and 0.08 (95% CI 0.07-0.09), respectively. When these types of NTDs are combined, Hispanic women had the highest overall rate (1.12, 95% CI 1.04-1.21), followed by Whites (0.96, 95% CI 0.89-1.04), Blacks (0.75, 95% CI 0.59-0.91), and Asians (0.75, 95% CI 0.60-0.90). Hispanic women were 45% more likely than White women to have a pregnancy affected with anencephaly (odds ratio = 1.45, 95% CI 1.24-1.70), while Asian women were over two times less likely to have a pregnancy affected with spina bifida (odds ratio = 0.44, 95% CI 0.29-0.65). Considerable variation exists in the prevalence of NTDs by race/ethnicity and by type of NTD, with Hispanic women exhibiting the highest overall NTD rate.

Universal newborn screening for Hb H disease in California.

Newborn screening is an accepted public health measure to ensure that appropriate health care is provided in a timely manner to infants with hereditary/metabolic disorders. Alpha-thalassemia is a common hemoglobin (Hb) disorder, and causes Hb H (beta4) disease, and usually fatal homozygous alpha(0)-thalassemia, also known as Hb Bart's (gamma4) hydrops fetalis syndrome. In 1996, the State of California began to investigate the feasibility of universal newborn screening for Hb H disease. Initial screening was done on blood samples obtained by heel pricks from newborns, and stored as dried blood spots on filter paper. Hb Bart's levels were measured as fast-moving Hb by automated high-performance liquid chromatography (HPLC) identical to that currently used in newborn screening for sickle cell disease. Subsequent confirmation of Hb H disease was done by DNA-based diagnostics for alpha-globin genotyping. A criterion of 25% or more Hb Bart's as determined by HPLC detects most, if not all cases of Hb H disease, and few cases of alpha-thalassemia trait. From January, 1998, through June, 2000, 89 newborns were found to have Hb H disease. The overall prevalence for Hb H disease among all newborns in California is approximately 1 per 15,000. Implementation of this program to existing newborn hemoglobinopathy screening in populations with significant proportions of southeast Asians is recommended. The correct diagnosis would allow affected infants to be properly cared for, and would also raise awareness for the prevention of homozygous alpha(0)-thalassemia or Hb Bart's hydrops fetalis syndrome.

Extremely high phenylalanine levels in a newborn on parenteral nutrition: phenylketonuria in the neonatal intensive care unit.

A 1890-g newborn on total parenteral nutrition (TPN) had phenylalanine levels reaching 4164 µM indicating phenylketonuria (PKU). Review of 64 PKU cases from the California Newborn Screening Program disclosed another newborn diagnosed while on TPN. Phenylalanine levels rose five times faster with TPN, as estimated from rates in these infants. Thus, TPN use is associated with very high phenylalanine levels in newborns with PKU. When starting TPN soon after birth (for example, on day 1), early detection of PKU-by newborn screening 12 to 24 h after infusions are begun-should be helpful in limiting exposures to toxic levels of phenylalanine.

Asian immigration and public health in California: thalassemia in newborns in California.

PURPOSE: This study reviews Asian immigration in California and the effect it has had on public health in the state in terms of genetic disease detection. This is documented in terms of the numbers of cases of thalassemia detected, including Hemoglobin (Hb) E/beta-thalassemia, beta-thalassemia major, and Hb H disease. PATIENTS AND METHODS: California has been screening all newborns for hemoglobinopathies since 1990 and tests approximately 530,000 newborns per year. Samples are collected on filter paper during the first I to 2 days of life and sent to one of eight contract laboratories. The screening methodology is cation exchange high-performance liquid chromatography. Confirmatory testing is performed at Children's Hospital Oakland hemoglobin laboratory using a variety of methods. RESULTS: Approximately five to seven cases each of Hb E/beta-thalassemia and beta-thalassemia major are detected annually. Most cases are of Southeast Asian origin. Prevalence rate of Hb E/beta-thalassemia among Southeast Asians is approximately 1 in 2,200 births. A pilot program for Hb H disease screening was successful and this disorder has now been incorporated in newborn screening, detecting approximately 40 cases per year. CONCLUSIONS: Increases in Asian immigration and births in the U.S., particularly California, have been dramatic during the past 10 years and have led to detection of previously rare diseases like Hb E/beta-thalassemia. It has also changed the way other thalassemic disorders are viewed, such as Cooley anemia, which previously affected mainly individuals of Mediterranean origin. Now, most affected patients are of Asian origin.

Birth prevalence of primary congenital hypothyroidism by sex and ethnicity.

Over 5 million infants have been screened for primary congenital hypothyroidism in California since 1980. This large number along with the multi-ethnic nature of California's population allows for a detailed analysis of the effects of ethnic origin, sex, and their interaction on birth prevalence. Sex is the most important factor, with at least a 2:1 (female:male) ratio across all major ethnic groups except blacks. The sex ratio among Hispanics is more striking; female cases outnumber male cases by a ratio of 3:1, and the birth prevalence for Hispanic females is 1 in 1886 births. Previously published rates for Asians and blacks are suspect because of small sample sizes, and Hispanic rates also may be misleading if sex is not taken into account. These factors are important when screening tests, such as the serum T4 test, are used as a statistical prescreening before thyroid stimulation hormone levels can be determined and before the influence of ethnic group and sex can be taken into account, because other factors may prevent high-risk groups (such as Hispanic females) from being declared positive.

Bilirubin binding by the fractionated alternate allelic components of heterozygous monkey albumin.

Analysis of bilirubin-binding parameters for purified albumin of nine rhesus monkeys heterozygous for albumin MacA and albumin MacB was performed after separating these two albumin forms by fast protein liquid chromatography (FPLC). The binding capacity (n) for MacA-enriched samples was lower than that for the MacB variant in eight of nine fraction pairs analyzed, while the affinity constant (K) was higher in all nine MacA-enriched samples. The values for n X K were higher in MacA-enriched samples in eight of nine pairs tested. These data, together with previous studies, geographic specificity of the MacB variant, and the presence of dietary competitors for the primary bilirubin binding site on the albumin molecule, suggest an evolutionary advantage for the MacB variant only in areas where the dietary competitors are present.

Distribution of hemoglobinopathy variants by ethnicity in a multiethnic state.

It has been well known for many years that Black Americans are at increased risk for sickle cell disease and that individuals of Mediterranean ancestry are at increased risk for thalassemias. Beyond this, however, complete assessments of the epidemiology of hemoglobinopathies in Americans have been constrained by lack of large enough sample size, incomplete diagnostic work or testing inadequacies, or missing or misleading ethnicity data. California began universal, mandatory screening of all infants born in the state in 1990, and by January 1996, had screened over 3.3 million infants of various ethnic backgrounds. New information is now available on groups at increased risk for disease, as well as carrier status. The overall prevalence of sickle cell disease (all types) remains high in Blacks at 1 per 396 births, but is lower than expected (from east coast/ Caribbean published data) for Hispanics at 1 m 36,000 births or from previously published data on other higher risk groups such as Middle Eastern (no cases in over 22,000 screens) or Asian Indian (1 case in 16,000 screens). The distribution of different types of sickle cell disease has also changed, with increased numbers of Hb SE disease resulting from multiethnic partnerships. Demographic trends in California have led to a major sift in the at-risk groups for major beta thalassemias, with the majority of cases detected in families of Asian, Southeast Asian, and Asian Indian ancestry. The "new" hemoglobinopathy condition in California, Hb E/Beta. Thalassemia, is found almost exclusively in Southeast Asians with a prevalence of 1 in every 2,600 births. Carrier conditions are found in virtually every ethnic category, with higher than expected rates in non-Hispanic Whites (1 per 600 births).

Risk factors for congenital hypothyroidism: an investigation of infant's birth weight, ethnicity, and gender in California, 1990-1998.

BACKGROUND: Approximately 85% of primary congenital hypothyroidism (CH) is sporadic and due to malformations of the thyroid gland. Past studies have reported an increased birth weight among infants with CH. We have attempted to replicate and expand these observations, examining the association between different birth weight categories and CH stratified by infant's sex. We have also examined the prevalence of CH by mother's age and infant's ethnicity, gender, and year of birth. METHODS: A cross-sectional study was conducted on 5, 049,185 infants screened by the statewide California Newborn Screening Program between 1990 and 1998, an estimated 98.6% of all newborns in the state. Dried blood spots from a heel stick were assayed for thyroxine (T4), and presumptive positives had follow-up assays of thyroid-stimulating hormone (TSH) to determine definite positives. RESULTS: A total of 1,806 cases of CH were identified. The following findings are unlikely to be due to chance. Compared with infants with birth weights of 3,000-3,499 g, infants weighing <2,000 g and those weighing >/=4,500 g had a twofold or greater increase in the prevalence of CH. This was not explained as a result of confounding by the infant's ethnicity or gender. Compared with whites, elevated prevalence rates were found in most ethnic groups, which include the following: Hispanics, Chinese, Vietnamese, Asian Indians, Filipinos, Middle Easterners, and Hawaiians. As reported previously, black infants had about one-third the prevalence rate of whites. We also observed the frequently described female preponderance of CH. The female excess was maintained at all birth weights, however it varied by infant's ethnicity. Trends in the prevalence of CH were not associated with mother's age or with the time interval between 1990 and 1998. CONCLUSIONS: We observed an increased risk of CH in both low-birth-weight (<2,000-g) and macrosomic (>/=4,500-g) infants. This U-shaped association has not been described in past studies. We have also expanded the previously described ethnic differences in CH risk to include ethnic groups not previously studied. The unique pattern of CH occurrence suggests that further studies to define modifiable risk factors may be useful.

Prenatal ultrasound findings in affected and unaffected pregnancies that are screen-positive for trisomy 18: the California experience.

This study investigated whether significant differences in ultrasound findings exist between trisomy 18 affected and unaffected pregnancies positive by serum screening. Ultrasound reports were reviewed for 335 screen-positive women. This represented 65% of all trisomy 18 screen-positive patients who had follow-up services at any of 117 Californian state-approved Prenatal Diagnosis Centers during a six-month period from November 1, 1995 to April 30, 1996. Ultrasound reports were available for 100% of trisomy 18 fetuses diagnosed during the six month period (n=23). Ultrasound findings were reported as normal in 35% of the fetuses affected with trisomy 18. The number and type of abnormalities observed in the affected and unaffected groups are described. When compared to unaffected cases, the trisomy 18 affected fetuses had a greater re-dating discrepancy on follow-up ultrasound evaluation and significantly lower femur length to biparietal diameter (FL/BPD) ratio measurements. We recommend that all women who are screen positive for trisomy 18 be encouraged to have amniocentesis, regardless of ultrasound findings, since affected fetuses may not be detected otherwise.

Detection of Hb E/beta-thalassemia versus homozygous EE using high-performance liquid chromatography results from newborns.

The influx of Southeast Asian immigrants into California over the past few years has resulted in a dramatic increase of Hb E disorders detected in newborn screening. Initial hemoglobin patterns of FE do not distinguish between homozygous EE, a benign state, and E/beta-thalassemia, a clinically significant disorder which is frequently transfusion-dependent. Since language and cultural customs frequently prevent parent testing which can rule out the thalassemic disorder, and diagnosis in the neonate is not possible by traditional red cell indices and is relatively expensive by DNA methodology, an alternate screening method is proposed. This study investigated the Hb F and Hb E relative percentages obtained in the newborn's high-performance liquid chromatography result, and found that the percentage of Hb E was markedly lower in neonates with Hb E/beta-thalassemia versus those which were homozygous EE. Likewise, the F/E ratios were different in the E/beta-thalassemia group versus the EE group. This analysis can at least minimize the number of DNA tests required, and with more E/beta-thalassemia case data, may prove to be a reliable substitute.

Beta-globin haplotypes from blood spots for follow-up of newborn hemoglobinopathy screening.

The inheritance of sickle-cell anemia upon the background of the major beta-globin gene cluster haplotypes has been associated with differing risks for major organ failure, and more recently with response to hydroxyurea treatment. Early identification of beta-globin haplotypes in individuals with sickle-cell anemia may be a clinically useful prognostic factor for severity of disease expression. This report describes the use of whole-blood spots on filter papers from newborn hemoglobinopathy screening for beta-globin gene cluster haplotyping by the polymerase chain reaction.