Analysis of age-related changes in psychosine metabolism in the human brain.

α-Synuclein aggregation has been linked to Gaucher's disease (GD) and Krabbe's disease (KD), lysosomal conditions affecting glycosphingolipid metabolism. α-Synuclein pathology has been directly attributed to the dysregulation of glycosphingolipids in both conditions, specifically to increased galactosylsphingosine (psychosine) content in the context of KD. Furthermore, the gene (GALC) coding for the psychosine degrading enzyme galactosylceramidase (GALC), has recently been identified as a risk loci for Parkinson's disease. However, it is unknown if changes in psychosine metabolism and GALC activity in the context of the aging human brain correlate with Parkinson's disease. We investigated psychosine accumulation and GALC activity in the aging brain using fresh frozen post-mortem tissue from Parkinson's (PD, n = 10), Alzheimer's (AD, n = 10), and healthy control patients (n = 9), along with tissue from neuropsychiatric patients (schizophrenia, bipolar disorder and depression, n = 15 each). An expanded mutational analysis of PD (n = 20), AD (n = 10), and healthy controls (n = 30) examined if PD was correlated with carriers for severe GALC mutations. Psychosine content within the cerebral cortex of PD patients was elevated above control patients. Within all patients, psychosine displayed a significant (p<0.05) and robust regional distribution in the brain with higher levels in the white matter and substantia nigra. A mutational analysis revealed an increase in the incidence of severe GALC mutations within the PD patient population compared to the cohorts of Alzheimer's patients and healthy controls tested. In addition to α-synuclein pathology identified in the KD brain, control patients identified as GALC mutational carriers or possessing a GALC pathogenic variant had evidence of α-synuclein pathology, indicating a possible correlation between α-synuclein pathology and dysregulation of psychosine metabolism in the adult brain. Carrier status for GALC mutations and prolonged exposure to increased psychosine could contribute to α-synuclein pathology, supporting psychosine metabolism by galactosylceramidase as a risk factor for Parkinson's disease.

Liquid Chromatography-Tandem Mass Spectrometry Assay of Leukocyte Acid α-Glucosidase for Post-Newborn Screening Evaluation of Pompe Disease.

BACKGROUND: Pompe disease (PD) is the first lysosomal storage disorder to be added to the Recommended Uniform Screening Panel for newborn screening. This condition has a broad phenotypic spectrum, ranging from an infantile form (IOPD), with severe morbidity and mortality in infancy, to a late-onset form (LOPD) with variable onset and progressive weakness and respiratory failure. Because the prognosis and treatment options are different for IOPD and LOPD, it is important to accurately determine an individual's phenotype. To date, no enzyme assay of acid α-glucosidase (GAA) has been described that can differentiate IOPD vs LOPD using blood samples. METHODS: We incubated 10 µL leukocyte lysate and 25 µL GAA substrate and internal standard (IS) assay cocktail for 1 h. The reaction was purified by a liquid-liquid extraction. The extracts were evaporated and reconstituted in 200 µL methanol and analyzed by LC-MS/MS for GAA activity. RESULTS: A 700-fold higher analytical range was observed with the LC-MS/MS assay compared to the fluorometric method. When GAA-null and GAA-containing fibroblast lysates were mixed, GAA activity could be measured accurately even in the range of 0%-1% of normal. The leukocyte GAA activity in IOPD (n = 4) and LOPD (n = 19) was 0.44-1.75 nmol · h-1 · mg-1 and 2.0-6.5 nmol · h-1 · mg-1, respectively, with no overlap. The GAA activity of pseudodeficiency patients ranged from 3.0-28.1 nmol · h-1 · mg-1, showing substantial but incomplete separation from the LOPD group. CONCLUSIONS: This assay allows determination of low residual GAA activity in leukocytes. IOPD, LOPD, and pseudodeficiency patients can be partially differentiated by measuring GAA using blood samples.

Newborn screening for SCID in New York State: experience from the first two years.

PURPOSE: To describe the process and assess outcomes for the first 2 years of newborn screening for severe combined immunodeficiency (SCID NBS) in New York State (NYS). METHODS: The NYS algorithm utilizes a first-tier molecular screen for TRECs (T-cell receptor excision circles), the absence of which is indicative of increased risk of immunodeficiency. RESULTS: During the first 2 years, 485,912 infants were screened for SCID. Repeat specimens were requested from 561 premature and 746 non-premature infants with low or borderline TRECs. A total of 531 infants were referred for diagnostic evaluation leading to identification of 10 infants with SCID and 87 with a clinically significant non-SCID abnormality based on flow cytometry or CBC results (positive predictive value 20.3 %). Nine infants were diagnosed with typical SCID and one with leaky SCID. SCID diagnoses included two patients with adenosine deaminase deficiency, three patients with typical and one with leaky IL2RG-related SCID, one patient with IL7Rα-related SCID, and three cases of typical SCID, etiology unknown. TRECs were undetectable in eight of the nine babies with typical SCID. Infants with other non-SCID conditions included 27 patients with a syndrome that included T-cell impairment, 18 of which had DiGeorge syndrome. Seventeen infants had T-cell impairment secondary to another clinically significant condition, and 13 were classified as 'other'. Among 30 infants classified as idiopathic T-cell lymphopenia, 11 have since resolved, and the remainder continues to be followed. One infant with undetectable TRECs had normal follow-up studies. Molecular studies revealed the presence of two changes in the infant's DNA. CONCLUSIONS: Overall, ten infants with SCID were identified during the first 2 years of screening in NYS, yielding an incidence of approximately 1 in 48,500 live births, which is consistent with the incidence observed by other states screening for SCID. The incidence of any clinically significant laboratory abnormality was approximately 1 in 5,000; both estimates are higher than estimates prior to the onset of newborn screening for SCID. Improvements to the NYS algorithm included the addition of a borderline category that reduced the proportion of infants referred for flow cytometric analysis, without decreasing sensitivity. We identified a large number of infants with abnormal TRECs and subsequent idiopathic T-cell lymphopenia. Long-term follow-up studies are needed to determine the prognosis and optimal treatment for this group of patients, some of whom may present with previously unrecognized, transient lymphopenia of infancy.

Cost-effective and scalable DNA extraction method from dried blood spots.

BACKGROUND: Dried blood spot (DBS) samples have been widely used in newborn screening (NBS) for the early identification of disease to facilitate the presymptomatic treatment of congenital diseases in newborns. As molecular genetics knowledge and technology progresses, there is an increased demand on NBS programs for molecular testing and a need to establish reliable, low-cost methods to perform those analyses. Here we report a flexible, cost-efficient, high-throughput DNA extraction method from DBS adaptable to small- and large-scale screening settings. METHODS: Genomic DNA (g.DNA) was extracted from single 3-mm diameter DBS by the sequential use of red cell lysis, detergent-alkaline, and acid-neutralizing buffers routinely used in whole blood and plant tissue DNA extractions. We performed PCR amplification of several genomic regions using standard PCR conditions and detection methods (agarose gel, melting-curve analysis, TaqMan-based assays). Amplicons were confirmed by BigDye® Terminator cycle sequencing and compared with reference sequences. RESULTS: High-quality g.DNA was extracted from hundreds of DBS, as proven by mutation detection of several human genes on multiple platforms. Manual and automated extraction protocols were validated. Quantification of g.DNA by Oligreen® fluorescent nucleic acid stain demonstrated a normal population distribution closely corresponding with white blood cell counts detected in newborn populations. CONCLUSIONS: High-quality, amplifiable g.DNA is extractable from DBSs. Our method is adaptable, reliable, and scalable to low- and high-throughput NBS at low cost ($0.10/sample). This method is routinely used for molecular testing in the New York State NBS program.

Prevalence of a loss-of-function mutation in the proton-coupled folate transporter gene (PCFT-SLC46A1) causing hereditary folate malabsorption in Puerto Rico.

OBJECTIVE: To determine whether subjects of Puerto Rican heritage are at increased risk for a specific mutation of the proton-coupled folate transporter (PCFT) causing hereditary folate malabsorption (HFM). STUDY DESIGN: Three percent of the births in Puerto Rico in 2005, with additional regional oversampling, were screened for the prevalence of the c.1082G>A; p.Y362_G389 del PCFT gene mutation. Six new subjects of Puerto Rican heritage with the clinical diagnosis of HFM were also assessed for this mutation. RESULTS: Six subjects of Puerto Rican heritage with the clinical diagnosis of HFM were all homozygous for the c.1082G>A; p.Y362_G389 del PCFT mutation. Three heterozygote carriers were identified from the 1582 newborn samples randomly selected from births in Puerto Rico in 2005. The carrier frequency for the mutated allele was 0.2% island-wide and 6.3% in Villalba. CONCLUSION: These findings are consistent with a common mutation in the PCFT gene causing HFM that has disseminated to Puerto Ricans who have migrated to mainland United States. Because prompt diagnosis and treatment of infants with HFM can prevent the consequences of this disorder, newborn screening should be considered in high-risk populations and physicians should be aware of its prevalence in infants of Puerto Rican ancestry.

Development of genomic reference materials for cystic fibrosis genetic testing.

The number of different laboratories that perform genetic testing for cystic fibrosis is increasing. However, there are a limited number of quality control and other reference materials available, none of which cover all of the alleles included in commercially available reagents or platforms. The alleles in many publicly available cell lines that could serve as reference materials have neither been confirmed nor characterized. The Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community as well as Coriell Cell Repositories, have characterized an extended panel of publicly available genomic DNA samples that could serve as reference materials for cystic fibrosis testing. Six cell lines [containing the following mutations: E60X (c.178G>T), 444delA (c.312delA), G178R (c.532G>C), 1812-1G>A (c.1680-1G>A), P574H (c.1721C>A), Y1092X (c.3277C>A), and M1101K (c.3302T>A)] were selected from those existing at Coriell, and seven [containing the following mutations: R75X (c.223C>T), R347H (c.1040G>A), 3876delA (c.3744delA), S549R (c.1646A>C), S549N (c.1647G>A), 3905insT (c.3773_3774insT), and I507V (c.1519A>G)] were created. The alleles in these materials were confirmed by testing in six different volunteer laboratories. These genomic DNA reference materials will be useful for quality assurance, proficiency testing, test development, and research and should help to assure the accuracy of cystic fibrosis genetic testing in the future. The reference materials described in this study are all currently available from Coriell Cell Repositories.

The effects of chemical and heat maceration techniques on the recovery of nuclear and mitochondrial DNA from bone.

Forensic anthropologists use a number of maceration techniques to facilitate skeletal analysis of personal identity and trauma, but they may unwittingly eliminate valuable DNA evidence in the process. This study evaluated the effect of 10 maceration methods on gross bone structure and the preservation of DNA in ribs of 12 pigs (Sus scrofa). A scoring system was applied to evaluate the ease of maceration and resulting bone quality while DNA purity was quantified by optical densitometry analysis, followed by polymerase chain reaction (PCR) amplification of three mitochondrial and three nuclear loci. The results demonstrated that while mitochondrial DNA could be amplified for all experiments, cleaning treatments using bleach, hydrogen peroxide, ethylenediaminetetraacetic acid/papain, room temperature water and detergent/sodium carbonate followed by degreasing had low DNA concentrations and failed to generate nuclear PCR products. In general, treatments performed at high temperatures (90 degrees C or above) for short durations performed best. This study shows that traditionally "conservative" maceration techniques are not necessarily the best methods to yield DNA from skeletal tissue.