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Despite substantial progress, causal variants are identified only for a minority of familial Parkinson's disease (PD) cases, leaving high-risk pathogenic variants unidentified 1,2 . To identify such variants, we uniformly processed exome sequencing data of 2,184 index familial PD cases and 69,775 controls. Exome-wide analyses converged on RAB32 as a novel PD gene identifying c.213C > G/p.S71R as a high-risk variant presenting in ~0.7% of familial PD cases while observed in only 0.004% of controls (odds ratio of 65.5). This variant was confirmed in all cases via Sanger sequencing and segregated with PD in three families. RAB32 encodes a small GTPase known to interact with LRRK2 (refs. 3,4 ). Functional analyses showed that RAB32 S71R increases LRRK2 kinase activity, as indicated by increased autophosphorylation of LRRK2 S1292. Here our results implicate mutant RAB32 in a key pathological mechanism in PD-LRRK2 kinase activity 5-7 -and thus provide novel insights into the mechanistic connections between RAB family biology, LRRK2 and PD risk., (© 2024. The Author(s).)

A decline in intellectual functioning (intelligence quotient [IQ]) is often observed following more severe forms of traumatic brain injury (TBI) and is a useful index for long-term outcome. Identifying brain correlates of IQ can serve to inform developmental trajectories of behavior in this population. Using magnetic resonance imaging (MRI), we examined the relationship between intellectual abilities and patterns of cortical thickness in children with a history of TBI or with orthopedic injury (OI) in the chronic phase of injury recovery. Participants were 47 children with OI and 58 children with TBI, with TBI severity ranging from complicated-mild to severe. Ages ranged from 8 to 14 years old, with an average age of 10.47 years, and an injury-to-test range of ∼1-5 years. The groups did not differ in age or sex. The intellectual ability estimate (full-scale [FS]IQ-2) was derived from a two-form (Vocabulary and Matrix Reasoning subtests) Wechsler Abbreviated Scale of Intelligence (WASI). MRI data were processed using the FreeSurfer toolkit and harmonized across data collection sites using neuroComBat procedures, while holding demographic features (i.e., sex, socioeconomic status [SES]), TBI status, and FSIQ-2 constant. Separate general linear models per group (TBI and OI) and a single interaction model with all participants were conducted with all significant results withstanding correction for multiple comparisons via permutation testing. Intellectual ability was higher ( p  < 0.001) in the OI group (FSIQ-2 = 110.81) than in the TBI group (FSIQ-2 = 99.81). In children with OI, bi-hemispheric regions, including the right pre-central gyrus and precuneus and bilateral inferior temporal and left occipital areas were related to IQ, such that higher IQ was associated with thicker cortex in these regions. In contrast, only cortical thickness in the right pre-central gyrus and bilateral cuneus positively related to IQ in children with TBI. Significant interaction effects were found in the bilateral temporal, parietal, and occipital lobes and left frontal regions, indicating that the relationship between IQ and cortical thickness differed between groups in these regions. Changes in cortical associations with IQ after TBI may reflect direct injury effects and/or adaptation in cortical structure and intellectual functioning, particularly in the bilateral posterior parietal and inferior temporal regions. This suggests that the substrates of intellectual ability are particularly susceptible to acquired injury in the integrative association cortex. Longitudinal work is needed to account for normal developmental changes and to investigate how cortical thickness and intellectual functioning and their association change over time following TBI. Improved understanding of how TBI-related cortical thickness alterations relate to cognitive outcome could lead to improved predictions of outcome following brain injury.

Studies show that maternal behaviors are mediated by the bivariate serotonin transporter (5-HTT) genotype, although the findings are mixed, with some studies showing that mothers with the s allele exhibit increased maternal sensitivity, while other studies show that mothers with the s allele show decreased maternal sensitivity. Nonhuman primate studies offer increased control over extraneous variables and may contribute to a better understanding of the effects of the 5-HTT genotype on maternal sensitivity. This study assesses the influence of 5-HTT genotype variation on maternal sensitivity in parenting in 125 rhesus macaque mothers (Macaca mulatta) during the first three-months of their infants' lives, an age well before typical infants undergo weaning. Mothers were genotyped for the 5-HTT genotype and maternal behaviors were collected, including neglectfulness, sensitivity, and premature rejections during undisturbed social interactions. Results showed that mothers homozygous for the s allele rejected their infants the most and restrained their infants the least, an indication that mothers with the s allele are more likely to neglect their infants' psychological and physical needs. These findings suggest that, at an age when an infant's needs are based on warmth, security, and protection, mothers with an s allele exhibit less sensitive maternal behaviors. High rates of rejections and low rates of restraints are behaviors that typically characterize premature weaning and are inappropriate for their infant's young age. This study is an important step in understanding the etiology of variability in maternal warmth and care, and further suggests that maternal 5-HTT genotype should be examined in studies assessing genetic influences on variation in maternal sensitivity, and ultimately, mother-infant attachment quality., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wood et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

ERAP1 is a key aminopeptidase involved in peptide trimming before major histocompatibility complex (MHC) presentation. A single nucleotide polymorphism (SNP) in the ERAP1 gene can lead to impaired trimming activity and affect ERAP1 function. ERAP1 genetic variations have been linked to an increased susceptibility to cancer and autoimmune disease. Here, we report the discovery of novel ERAP1 inhibitors using a high throughput screening approach. Due to ERAP1 broad substrate specificity, the hit finding strategy included testing inhibitors with a range of biochemical assays. Based on the hit potency, selectivity, and in vitro absorption, distribution, metabolism, excretion, and toxicity, the benzofuran series was selected. Fifteen derivatives were designed and synthesized, the compound potency was improved to the nanomolar range, and the structure-activity relationship supported by modeling studies., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

We have used SWATH mass spectrometry to quantify 3648 proteins across 76 proteomes collected from genetically diverse BXD mouse strains in two fractions (mitochondria and total cell) from five tissues: liver, quadriceps, heart, brain, and brown adipose (BAT). Across tissues, expression covariation between genes' proteins and transcripts-measured in the same individuals-broadly aligned. Covariation was however far stronger in certain subsets than others: only 8% of transcripts in the lowest expression and variance quintile covaried with their protein, in contrast to 65% of transcripts in the highest quintiles. Key functional differences among the 3648 genes were also observed across tissues, with electron transport chain (ETC) genes particularly investigated. ETC complex proteins covary and form strong gene networks according to tissue, but their equivalent transcripts do not. Certain physiological consequences, such as the depletion of ATP synthase in BAT, are thus obscured in transcript data. Lastly, we compared the quantitative proteomic measurements between the total cell and mitochondrial fractions for the five tissues. The resulting enrichment score highlighted several hundred proteins which were strongly enriched in mitochondria, which included several dozen proteins were not reported in literature to be mitochondrially localized. Four of these candidates were selected for biochemical validation, where we found MTAP, SOAT2, and IMPDH2 to be localized inside the mitochondria, whereas ABCC6 was in the mitochondria-associated membrane. These findings demonstrate the synergies of a multi-omics approach to study complex metabolic processes, and this provides a resource for further discovery and analysis of proteoforms, modified proteins, and protein localization., (© 2018 Williams et al.)

There have been many attempts to unveil the therapeutic potential of antisense molecules during the last decade. Due to its specific role in canonical Wnt signalling, β-catenin is a potential target for an antisense-based antitumour therapy. In order to establish such a strategy with peptide nucleic acids, we developed a reporter assay for quantification of antisense effects. The luciferase-based assay detects splice blocking with high sensitivity. Using this assay, we show that the splice donor of exon 13 of β-catenin is particularly suitable for an antisense strategy, as it results in a truncated protein which lacks transactivating functions. Since the truncated proteins retain the interactions with Tcf/Lef proteins, they act in a dominant negative fashion competing with wild-type proteins and thus blocking the transcriptional activity of β-catenin. Furthermore, we show that the truncation does not interfere with binding of cadherin and α-catenin, both essential for its function in cell adhesion. Therefore, the antisense strategy blocks Wnt signalling with high efficiency but retains other important functions of β-catenin.

The LRRK2 G2019S mutation is found at higher frequency among Parkinson disease (PD) patients of Ashkenazi Jewish (AJ) ancestry. This study was designed to test whether an internet-based approach could be an effective approach to screen and identify mutation carriers. Individuals with and without PD of AJ ancestry were recruited and consented through an internet-based study website. An algorithm was applied to a series of screening questions to identify individuals at increased risk to carry the LRRK2 G2019S mutation. About 1000 individuals completed the initial screening. Around 741 qualified for mutation testing and 650 were tested. Seventy-two individuals carried at least one LRRK2 G2019S mutation; 38 with PD (12.5%) and 34 without (10.1%). Among the AJ PD participants, each affected first-degree relative increased the likelihood the individual was LRRK2+ [OR = 4.7; 95% confidence interval = (2.4-9.0)]. The same was not observed among the unaffected AJ subjects (P = 0.11). An internet-based approach successfully screened large numbers of individuals to identify those with risk factors increasing the likelihood that they carried a LRRK2 G2019S mutation. A similar approach could be implemented in other disorders to identify individuals for clinical trials, biomarker analyses and other types of research studies.

Background: Red cell (RBC) blood group alloimmunization remains a major problem in transfusion medicine. Patients with sickle cell disease (SCD) are at particularly high risk for developing alloantibodies to RBC antigens compared to other multiply transfused patient populations. Hemagglutination is the classical method used to test for blood group antigens, but depending on the typing methods and reagents used may result in discrepancies that preclude interpretation based on serologic reactivity alone. Molecular methods, including customized DNA microarrays, are increasingly used to complement serologic methods in predicting blood type. The purpose of this study was to determine the diversity and frequency of RH alleles in African Americans and to assess the performance of a DNA microarray for RH allele determination., Material and Methods: Two sets of samples were tested: (i) individuals with known variant Rh types and (ii) randomly selected African American donors and patients with SCD. Standard hemagglutination tests were used to establish the Rh phenotype, and cDNA- and gDNA-based analyses (sequencing, PCR-RFLP, and customized RHD and RHCE microarrays were used to predict the genotype)., Results: In a total of 829 samples (1658 alleles), 72 different alleles (40 RHD and 32 RHCE) were identified, 22 of which are novel. DNA microarrays detected all nucleotides probed, allowing for characterization of over 900 alleles., Conclusions: High-throughput DNA testing platforms provide a means to test a relatively large number of donors and potentially prevent immunization by changing the way antigen-negative blood is provided to patients. Because of the high RH allelic diversity found in the African American population, determination of an accurate Rh phenotype often requires DNA testing, in conjunction with serologic testing. Allele-specific microarrays offer a means to perform high-throughput donor Rh typing and serve as a valuable adjunct to serologic methods to predict Rh type. Because DNA microarrays test for only a fixed panel of allelic polymorphisms and cannot determine haplotype phase, alternative methods such as Next Generation Sequencing hold the greatest potential to accurately characterize blood group phenotypes and ameliorate the clinical course of multiply-transfused patients with sickle cell disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: RHCE*ceMO has nucleotide changes 48G>C and 667G>T, which encode, respectively, 16Cys and 223Phe associated with altered expression of e antigen. RHD*DAU0 has Nucleotide 1136C>T, which encodes 379Met associated with normal levels of D. We compiled serologic and DNA testing data on samples with RHCE*ceMO to determine the red blood cell (RBC) antigen expression, antibody specificity, RHD association, and the prevalence in African-American persons., Study Design and Methods: Serologic testing was performed by standard methods. Genomic DNA was used for polymerase chain reaction-restriction fragment length polymorphism and RH-exon sequencing, and for some, Rh-cDNA was sequenced. Seventy-seven (50 donor and 27 patient) samples with RHCE*ceMO were studied, and 350 African-American persons were screened for allele prevalence., Results: RBCs from RHCE*ceMO homozygotes (or heterozygotes with RHCE*cE in trans) were weak or nonreactive with some anti-e and were nonreactive with polyclonal anti-hr(S) and anti-hr(B) . Twenty-three transfused patients homozygous for RHCE*ceMO/ceMO or with RHCE*ceMO in trans to RHCE*cE or *ce had alloanti-e, anti-f, anti-hr(S) /hr(B) , or an antibody to a high-prevalence Rh antigen. Three patients with alloanti-c had RHCE*ceMO in trans to RHCE*Ce. RHD*DAU0 was present in 30% of African-American persons tested and in 69 of 77 (90%) of samples with RHCE*ceMO., Conclusions: RHCE*ceMO encodes partial e, as previously reported, and also encodes partial c, a hr(S) - and hr(B) - phenotype, and the absence of a high-prevalence antigen (RH61). The antibody in transfused patients depended on the RHCE allele in trans. RHCE*ceMO was present in one in 50 African-American persons with an allele frequency of 0.01, is often linked to RHD*DAU0, and is potentially of clinical significance for transfusion., (© 2013 American Association of Blood Banks.)

Background: Diffusion of small molecules into fish embryos is essential for many experimental procedures in developmental biology and toxicology. Since we observed a weak uptake of lithium into medaka eggs we started a detailed analysis of its diffusion properties using small fluorescent molecules., Results: Contrary to our expectations, not the rigid outer chorion but instead membrane systems surrounding the embryo/yolk turned out to be the limiting factor for diffusion into medaka eggs. The consequence is a bi-phasic uptake of small molecules first reaching the pervitelline space with a diffusion half-time in the range of a few minutes. This is followed by a slow second phase (half-time in the range of several hours) during which accumulation in the embryo/yolk takes place. Treatment with detergents improved the uptake, but strongly affected the internal distribution of the molecules. Testing electroporation we could establish conditions to overcome the diffusion barrier. Applying this method to lithium chloride we observed anterior truncations in medaka embryos in agreement with its proposed activation of Wnt signalling., Conclusions: The diffusion of small molecules into medaka embryos is slow, caused by membrane systems underneath the chorion. These results have important implications for pharmacologic/toxicologic techniques like the fish embryo test, which therefore require extended incubation times in order to reach sufficient concentrations in the embryos.

Background: In the Rh blood group system, variant RhD and RhCE express several partial antigens. We investigated RH in samples with partial DIVa that demonstrated weak and variable reactivity with anti-C., Study Design and Methods: Standard hemagglutination techniques, polymerase chain reaction-based assays, and RH sequencing were used., Results: DNA analysis showed that six red blood cell (RBC) samples with weak and inconsistent reactivity with anti-C lacked RHCE*C, but all had RHD*DIVa, which encodes partial D and Go(a) . We then tested RBCs from 19 Go(a+) cryopreserved samples (confirmed to have RHD*DIVa) with four anti-C and observed weak variable reactions. RHCE genotyping found all but one of the samples with RHD*DIVa also had RHCE nt 48G>C and 1025C>T, named RHCE*ceTI. Lookback of samples referred for workup and found to have either allele revealed 47 of 55 had both RHD*DIVa and RHCE*ceTI, four had RHD*DIVa without RHCE*ceTI, and four had RHCE*ceTI without RHD*DIVa. Alloanti-c was found in a patient with c+ RBCs and RHCE*ceTI in trans to RHCE*Ce, and alloanti-e was found in a patient with e+ RBC and RHCE*ceTI in trans to RHCE*cE. RHD*DIVa in trans to RHD erroneously tested as RHD hemizygous., Conclusions: RHD*DIVa and RHCE*ceTI almost always, but not invariably, travel together. This haplotype is found in people of African ancestry and the RBCs can demonstrate aberrant reactivity with anti-C. RHCE*ceTI encodes partial c and e antigens. We confirm that RHD zygosity assays are unreliable in samples with RHD*DIVa., (© 2012 American Association of Blood Banks.)

Background: STEM (RH49) is a low-prevalence antigen in the Rh blood group system. A scarcity of anti-STEM has precluded extensive study of this antigen. We report that two alleles with a RHCE*ce818C>T change encode a partial e, and a hr(S) -, hr(B) +, STEM+ phenotype and that both alleles are frequently in cis to RHD*DOL1 or RHD*DOL2., Study Design and Methods: Blood samples were from donors and patients in our collections. Hemagglutination, DNA, and RNA testing was performed by standard techniques., Results: Fourteen STEM+ samples were heterozygous RHCE*ce818C/T: six had RHCE*ceBI and eight had a novel allele, RHCE*ceSM. Eleven were heterozygous for RHD*DOL1 or RHD*DOL2. Eleven samples, previously typed STEM-, had RHCE*ce818C/C (consensus nucleotide). RBCs from informative STEM+ samples were e+/- hr(S) - hr(B) +. One person who was heterozygous RHCE*ceBI and RHCE*cE had an anti-e-like antibody in her plasma, and one person, who was hemizygous for RHD*DOL2, had anti-D in her plasma., Conclusions: We show that two alleles with a RHCE*ce818C>T change (RHCE*ceBI and RHCE*ceSM) encode a hr(S) - hr(B) + STEM+ phenotype. In addition, both alleles are frequently in cis to RHD*DOL1 or RHD*DOL2 and RHCE*ceBI encodes a partial e antigen. In the small cohort of samples tested, RHD*DOL invariably traveled with RHCE*ce818T. Our study also confirmed the presumption that RHD*DOL2, like RHD*DOL1, encodes a partial D antigen and the low-prevalence antigen DAK., (© 2012 American Association of Blood Banks.)

Cell and gene therapies have an enormous range of potential applications, but as for most other therapies, dosing is a critical issue, which makes regulated gene expression a prerequisite for advanced strategies. Several inducible expression systems have been established, which mainly rely on small molecules as inducers, such as hormones or antibiotics. The application of these inducers is difficult to control and the effects on gene regulation are slow. Here we describe a novel system for induction of gene expression in encapsulated cells. This involves the modification of cells to express potential therapeutic genes under the control of a heat inducible promoter and the co-encapsulation of these cells with magnetic nanoparticles. These nanoparticles produce heat when subjected to an alternating magnetic field; the elevated temperatures in the capsules then induce gene expression. In the present study we define the parameters of such systems and provide proof-of-principle using reporter gene constructs. The fine-tuned heating of nanoparticles in the magnetic field allows regulation of gene expression from the outside over a broad range and within short time. Such a system has great potential for advancement of cell and gene therapy approaches., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Due to their homology, close proximity, and opposite orientation, RHD and RHCE can exchange nucleotides giving rise to variant alleles. Some of these variants encode the so-called partial phenotypes. The DIII partial D category has been subdivided into DIIIa, DIIIb, DIIIc, DIII Type 4, DIII Type 6, and DIII Type 7. During DNA-based screening tests, we identified a second example of DIII Type 7 in a Dce donor from South Africa. Our study describes hemagglutination tests on this sample and raises a question regarding the molecular basis of the originally defined DIIIb category., Study Design and Methods: Hemagglutination and DNA testing were performed by standard techniques., Results: Red blood cells from this DIII Type 7 donor typed D+C-E-c+e+G-, DAK+ and did not react with anti-D made by people with the DIII phenotype. The allele is RHD*DIII 150C, 178C, 201A, 203A, 307C, 410T, 455C, 602G, 667G., Conclusions: Based on the serotype and ethnicity (black African), it is likely that DIII Type 7 is the originally defined DIIIb category., (© 2011 American Association of Blood Banks.)

Mutations in the leucine-rich repeat kinase 2 gene, located at 12q12, are the most common known genetic causes of Parkinson's disease. Studies of leucine-rich repeat kinase 2 mutation carriers have shown incomplete and age-dependent penetrance, and previous studies have suggested that inherited susceptibility factors may modify the penetrance of leucine-rich repeat kinase 2 mutations. Genomewide linkage to age of onset of leucine-rich repeat kinase 2-related Parkinson's disease was evaluated in a sample of 113 leucine-rich repeat kinase 2 mutation carriers from 64 families using single-nucleotide polymorphism data from the Illumina HumanCNV370 genotyping array. Association between onset age and single-nucleotide polymorphisms under suggestive linkage peaks was also evaluated. The top logarithmic odds score for onset age (logarithmic odds score = 2.43) was in the chromosome 1q32.1 region. Moderate linkage to onset was also identified at 16q12.1 (logarithmic odds score = 1.58). Examination of single-nucleotide polymorphism association to Parkinson's disease onset under the linkage peaks revealed no statistically significant single-nucleotide polymorphism associations. The 2 novel genomic regions identified may harbor modifiers of leucine-rich repeat kinase 2-related Parkinson's disease onset age or penetrance, and further study of these regions may provide important insight into leucine-rich repeat kinase 2-related Parkinson's disease., (Copyright © 2011 Movement Disorder Society.)

Background: Heavy alcohol consumption may accelerate the progression of hepatitis C-related liver disease and/or limit efforts at antiviral treatment in opioid-dependent patients receiving heroin-assisted treatment (HAT). Our study aims to assess alcohol intake among HAT patients by self-reports compared to direct ethanol metabolites., Method: Fifty-four patients in HAT were recruited from the centre for HAT at the University of Basel, Switzerland. The patients completed the Alcohol Use Disorder Identification Test (AUDIT), a self-report questionnaire on past-week ethanol intake and provided samples for the determination of ethyl glucuronide (UEtG) and ethyl sulphate (UEtS) in urine and of ethyl glucuronide (HEtG) in hair., Results: Eighteen patients scored above the AUDIT cut-off levels. Twenty-six patients tested positive for UEtG and 29 for UEtS. HEtG identified ethanol intake of more than 20 g/d in 20 additional cases that did not appear in the AUDIT. Using the total score of the AUDIT, HEtG detected 14 additional cases of relevant alcohol intake., Conclusions: The findings of this study, which is the first assessing alcohol intake in HAT patients using direct ethanol metabolites and self reports, suggest the complementary use of both. Improved detection of hazardous or harmful alcohol consumption in the context of HCV and heroin dependence will allow for earlier intervention in this population. This ultimately will contribute to an improvement in quality of life of patients in HAT. Furthermore, a significant reduction of costs can be achieved through a reduction of complications caused by alcohol intake., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The alleles RHCE*ceBI (RHCE*ce 48C, 712G, 818T, 1132G) and RHCE*ceSM (RHCE*ce 48C, 712G, 818T) encode the low-prevalence Rh antigen STEM. These alleles frequently travel in cis with RHD*DOL. To estimate the frequency of these alleles, we tested a total of more than 700 samples in two populations. Blood samples were obtained from patients with sickle cell disease and from blood donors of African descent. DNA extractions and analyses were performed by standard methods. In the United States, none of 70 patient samples had the RHCE*818 nucleotide change. Two of 220 donors (frequency of 0.009) were heterozygous for RHCE*818C/T (RHCE*ceBI). One of these samples had RHD/RHD*DOL and the other had RHD/RHD*DOL-2. In these 290 samples, no other RHD*DOL alleles were found. In Brazil, 1 of 244 patients with sickle cell disease (frequency of 0.004) and 1 of 171 donors (frequency of 0.006) were heterozygous for RHCE*818C/T (RHCE*ceBI). Testing of more than 500 additional samples from people of African descent, selected because they had a diverse range of common and variant RHCE alleles, did not reveal a sample with RHD*DOL or RHD/RHD*DOL-2 in the absence of RHCE*ce(818T). Although the numbers are small, our study shows that in the United States, the frequency of RHCE*818T is 0.007 (2 in 290 samples) and in Brazil it is 0.004 (2 in 515 samples). The four RHCE*818T alleles were RHCE*ceBI.

Copy number variants (CNVs) are known to cause Mendelian forms of Parkinson disease (PD), most notably in SNCA and PARK2. PARK2 has a recessive mode of inheritance; however, recent evidence demonstrates that a single CNV in PARK2 (but not a single missense mutation) may increase risk for PD. We recently performed a genome-wide association study for PD that excluded individuals known to have either a LRRK2 mutation or two PARK2 mutations. Data from the Illumina370Duo arrays were re-clustered using only white individuals with high quality intensity data, and CNV calls were made using two algorithms, PennCNV and QuantiSNP. After quality assessment, the final sample included 816 cases and 856 controls. Results varied between the two CNV calling algorithms for many regions, including the PARK2 locus (genome-wide p = 0.04 for PennCNV and p = 0.13 for QuantiSNP). However, there was consistent evidence with both algorithms for two novel genes, USP32 and DOCK5 (empirical, genome-wide p-values<0.001). PARK2 CNVs tended to be larger, and all instances that were molecularly tested were validated. In contrast, the CNVs in both novel loci were smaller and failed to replicate using real-time PCR, MLPA, and gel electrophoresis. The DOCK5 variation is more akin to a VNTR than a typical CNV and the association is likely caused by artifact due to DNA source. DNA for all the cases was derived from whole blood, while the DNA for all controls was derived from lymphoblast cell lines. The USP32 locus contains many SNPs with low minor allele frequency leading to a loss of heterozygosity that may have been spuriously interpreted by the CNV calling algorithms as support for a deletion. Thus, only the CNVs within the PARK2 locus could be molecularly validated and associated with PD susceptibility.

Background: The partial D phenotype DIIIa was originally reported to be associated with 455A>C in Exon 3, 602C>G in Exon 4, and 667T>G in Exon 5. Other alleles with these changes were subsequently identified and designated DIII Types 5, 6, and 7, as they had additional alterations. The observation that DNA samples associated with the DIIIa phenotype had more changes than those originally reported motivated us to reanalyze the DIIIa probands (BP and DJ) from the original study. We also studied additional DIIIa samples to clarify the RHD background and establish the associated RHCE., Study Design and Methods: Hemagglutination testing was performed by standard methods. RHD and RHCE were analyzed by combinations of polymerase chain reaction-restriction fragment length polymorphism, exon-specific sequencing, cloning, or direct sequencing of Rh-cDNAs., Results: The RHD alleles from BP, DJ, and 58 additional DIIIa samples had the three reported nucleotide changes as well as 186G>T, 410C>T, and 819G>A. The DIIIa allele was associated with several altered RHCE*ce-alleles, the prominent one being ceS (48C, 733G, 1006T)., Conclusion: The DIIIa phenotype is associated with six RHD changes, five of which encode amino acid changes, and partial DIIIa and DIII Type 5 are encoded by the same RHD allele. In all samples, RHD*DIIIa was inherited with altered RHCE*ce. Patients with partial DIIIa are at risk for production of alloanti-D, but they are also at risk for alloanti-e, -c, or antibodies to high-prevalence Rh antigens if there is no conventional RHCE*ce in trans. Among 39 patients studied, 16 had alloanti-D and 27 had alloanti-e or anti-hrB.

New treatments of type 2 diabetes have been developed, especially with the use of the properties of incretins, gastrointestinal hormones involved in glucose homeostasis. GLP-1 is responsible for most incretin effect with a rate that increases within minutes after meal intake, suggesting that its secretion is initially triggered by the combination of endocrine and nervous signals. The effects of GLP-1 on insulin secretion and glucagon are observed only at high glucose levels (glucose-dependent effects). In the type 2 diabetes, the incretin effect is altered due to reduced plasma concentrations of GLP-1, but its activity is intact. There are two innovative therapeutic approaches aimed to restore the incretin effect in patients with type 2 diabetes: the agonists of GLP-1 receptors administered subcutaneously that replace the deficiency of GLP-1; and the DPP-4 inhibitors that can prolong the life of endogenous GLP-1 by reducing activity of the enzyme DPP-4 that degrades GLP-1, molecules offering the advantage of oral administration. Their effectiveness on the glucose metabolism is around 0.5 to 1.1% and 0.8 to 1.5% in reduction in HbAlc for DPP-4 inhibitors and agonists of GLP-1 receptors, respectively. Moreover these latter have extra pancreatic effects, particularly by reducing gastric emptying and control of satiety, resulting in a weight loss of 1.6 to 3.8 kg. Their tolerance is generally good especially for the DPP-4 inhibitors, whereas agonists in GLP-1 receptor often cause nausea or vomiting at the initiation of the therapy. However their effectiveness and long-term safety need to be evaluated.

Thr Rh blood group system is highly complex both in the number of discreet antigens and in the existence of partial antigens, especially D and e. Recently, several partial c antigens have been reported. Here we report findings on an African American man with sickle cell disease whose RBCs typed C+c+ and whose plasma contained anti-c. Hemagglutination tests, DNA extraction, PCR-RFLP, reticulocyte RNA isolation, RT-PCR cDNA analyses, cloning, and sequencing were performed by standard procedures. RBCs from the patient typed C+c+ but his plasma contained alloanti-c. DNA analyses showed the presence of RHCE*Ce in trans to RHCE*ceAR with RHD*D and RHD*Weak D type 4.2.2. The amino acid changes on RhceAR are such that C+c+ patient made alloanti-c. This case shows that RhceAR carries a partial c antigen and illustrates the value of DNA testing as an adjunct to hemagglutination to aid in antibody identification in unusual cases.

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) library which contains over 5,600 spectra of 1,253 compounds relevant in clinical and forensic toxicology has been developed using a hybrid tandem mass spectrometer with a linear ion trap. Pure compound solutions-in some cases solutions made of tablets-were prepared and 1 to 2,000 ng of each compound were injected into the system using standard reversed-phase analytical columns with gradient elution. To obtain maximum mass spectral information enhanced product ion spectra were acquired with positive and/or negative ionization at low, medium, and high collision energies and additionally applying collision energy spread. In this mode, all product ions generated by the different collision energies are trapped in the linear ion trap prior to their detection. The applicability of the library for other types of hybrid tandem mass spectrometers with a linear ion trap of the same manufacturer as well as a standard triple-quadrupole tandem mass spectrometer has been investigated with a selection of compounds. The spectra of the developed library can be used to create methods for target analysis, either screening methods or quantitative procedures by generating transitions for multiple reaction monitoring. For those procedures, suitable transitions and convenient collision energies are selected from the library. It also has been utilized to identify compounds with a multi target screening approach for clinical and forensic toxicology with a standardized and automated system. The novel aspects compared to our former library produced with a standard triple-quadrupole mass spectrometer are the enlargement of the ESI-MS/MS library and the additional acquisition of spectra with collision energy spread.

Background: Age at onset in Parkinson disease (PD) is a highly heritable quantitative trait for which a significant genetic influence is supported by multiple segregation analyses. Because genes associated with onset age may represent invaluable therapeutic targets to delay the disease, we sought to identify such genetic modifiers using a genomewide association study in familial PD. There have been previous genomewide association studies (GWAS) to identify genes influencing PD susceptibility, but this is the first to identify genes contributing to the variation in onset age., Methods: Initial analyses were performed using genotypes generated with the Illumina HumanCNV370Duo array in a sample of 857 unrelated, familial PD cases. Subsequently, a meta-analysis of imputed SNPs was performed combining the familial PD data with that from a previous GWAS of 440 idiopathic PD cases. The SNPs from the meta-analysis with the lowest p-values and consistency in the direction of effect for onset age were then genotyped in a replication sample of 747 idiopathic PD cases from the Parkinson Institute Biobank of Milan, Italy., Results: Meta-analysis across the three studies detected consistent association (p < 1 x 10(-5)) with five SNPs, none of which reached genomewide significance. On chromosome 11, the SNP with the lowest p-value (rs10767971; p = 5.4 x 10(-7)) lies between the genes QSER1 and PRRG4. Near the PARK3 linkage region on chromosome 2p13, association was observed with a SNP (rs7577851; p = 8.7 x 10(-6)) which lies in an intron of the AAK1 gene. This gene is closely related to GAK, identified as a possible PD susceptibility gene in the GWAS of the familial PD cases., Conclusion: Taken together, these results suggest an influence of genes involved in endocytosis and lysosomal sorting in PD pathogenesis.

Background: The determination of blood group antigens in patients and donors is of primary importance in transfusion medicine. Blood group antigens are inherited and are polymorphic in nature. The majority of polymorphic blood group antigens arise from single-nucleotide polymorphisms (SNPs) in the blood group genes. Many DNA-based assays, such as species-specific polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism, and microchips, have been described to study variant blood group genes. In this study, the SNaPshot (Applied Biosystems) method was adapted to detect SNPs in 10 common blood group systems., Study Design and Methods: DNA regions of interest were amplified in multiplex PCR and annealed to specific oligonucleotide probe primers of different lengths. AmpliTaq DNA polymerase extended the primers by adding only a single fluorescent ddNTP to its 3' end and was detected by differential mobility in capillary electrophoresis in a genetic analyzer. Results were analyzed using computer software in SNaPshot default analysis method., Results: Seventeen SNP sites in 29 blood samples, previously phenotyped and/or genotyped, were used to test the accuracy and reproducibility of multiplex SNaPshot assays. The results were compared with the previously analyzed types. SNaPshot analyses predicted the 17 SNP sites accurately for all the 29 blood samples. Both homozygous and heterozygous blood groups were detected with equal confidence., Conclusion: Blood group detection by SNaPshot method is a practical alternative to antibody-dependent phenotype prediction. Starting with DNA, this method is fast with a turnaround time of 24 hours with mean reagent cost around $2 per SNP detected.

Five genes have been identified that contribute to Mendelian forms of Parkinson disease (PD); however, mutations have been found in fewer than 5% of patients, suggesting that additional genes contribute to disease risk. Unlike previous studies that focused primarily on sporadic PD, we have performed the first genomewide association study (GWAS) in familial PD. Genotyping was performed with the Illumina HumanCNV370Duo array in 857 familial PD cases and 867 controls. A logistic model was employed to test for association under additive and recessive modes of inheritance after adjusting for gender and age. No result met genomewide significance based on a conservative Bonferroni correction. The strongest association result was with SNPs in the GAK/DGKQ region on chromosome 4 (additive model: p = 3.4 x 10(-6); OR = 1.69). Consistent evidence of association was also observed to the chromosomal regions containing SNCA (additive model: p = 5.5 x 10(-5); OR = 1.35) and MAPT (recessive model: p = 2.0 x 10(-5); OR = 0.56). Both of these genes have been implicated previously in PD susceptibility; however, neither was identified in previous GWAS studies of PD. Meta-analysis was performed using data from a previous case-control GWAS, and yielded improved p values for several regions, including GAK/DGKQ (additive model: p = 2.5 x 10(-7)) and the MAPT region (recessive model: p = 9.8 x 10(-6); additive model: p = 4.8 x 10(-5)). These data suggest the identification of new susceptibility alleles for PD in the GAK/DGKQ region, and also provide further support for the role of SNCA and MAPT in PD susceptibility.

This study was conducted to identify the alcohol consumption among hepatitis C-positive people receiving opioid maintenance therapy using self-report and biomarkers. A total of 49 people (28 male, 21 female) were hepatitis C virus (HCV) positive and were included. The alcohol use disorder identification test (AUDIT) and self-reported ethanol intake in the last 28 days were assessed. In addition to gamma-glutamyl-transferase (GGT) and mean corpuscular volume (MCV), ethyl glucuronide (EtG) and ethyl sulphate (EtS) were determined in serum and urine (UEtG, UEtS, SEtG) using liquid chromatography/tandem mass-spectroscopy (LC/MS-MS) with deuterated internal standards. Abstinence from alcohol was reported for the last 28 days by 13 participants and for the last 7 days by 22. AUDIT was > 8 in 27 cases. The maximum values were 34.8 mg/l for UEtG, 5.3 mg/l for UEtS and 0.15 for SEtG. Among the 19 UEtG positives, 8 had not reported any ethanol intake in the 7 days prior to the study. Six participants reported intake of up to 320 g of ethanol in the last 7 days, but were negative for SEtG, UEtG and UEtS. Self-reported ethanol intake in the last 28 days correlated with AUDIT score (r = 0.733, P < 0.001), with the direct ethanol metabolites and MCV. In this population, abstinence and episodic heavy drinking are more common than in the general population. Episodic heavy drinking is a significant cause of acute risk in this population. Results from biomarker testing could indicate cases of under- as well as over-reporting of alcohol consumption. Further research on the diagnostic accuracy of direct ethanol metabolites, including the use of phosphatidylethanol (PEth), in this setting is needed.

Background: Heavy alcohol consumption may accelerate the progression of hepatitis C (HCV)-related liver disease and/or limit efforts at antiviral treatment. As most of the patients in methadone maintenance treatment (MMT) suffer from hepatitis C infection, this study was conducted to identify the alcohol intake among these patients at a Swiss Psychiatric University Clinic by self-reports and direct ethanol metabolites as biomarkers of ethanol consumption., Patients and Methods: A convenience sample of 40 MMT patients (15 women, 25 men; median age 39 years) of the total 124 patients was asked and consented to participate in this study. This sample was not different in age, gender distribution, and rate of hepatitis C infection from the total sample. The Alcohol Use Disorders Identification Test (AUDIT) and self-reported ethanol intake during the previous 7 days were assessed. In addition, ethyl glucuronide (EtG) in urine, and fatty acid ethyl esters (FAEEs) and EtG in hair were determined using LC-MS/MS and gas chromatograph/mass spectrometer. The limit of quantitation for UEtG, HEtG, and FAEEs were 0.1 mg/l, 2.3 pg/mg, and 0.1 ng/mg, respectively., Results: Fourteen participants reported abstinence from alcohol for the previous 7 days. AUDIT scores were > or =8 in 15 male and >5 in 5 female participants. Direct ethanol metabolites were as follows (median, min, max, standard deviation): UEtG (19 positives; 9.91, 1.38 to 251, 62.39 mg/l); the values of HEtG were 17.65, 0 to 513, 105.62 pg/mg [in 2 cases no material, 8 abstinent (up to 7 pg/mg), 15 social drinkers (up to 50 g per day), and 15 excessive users (>50/60 g/d)]. For the 13 cases, where enough material for additional determination of HFAEEs was available, the values were 0.32, 0 to 1.32, 0.44 ng/mg. Among the 30 HEtG-positive participants, 20 had not reported the corresponding ethanol intake using question 1 (frequency) and 2 (quantity) of the AUDIT. Of the 14 participants reporting no alcohol intake during the previous 7 days, 4 were UEtG-positive. HEtG and AUDIT correlated significantly (r = 0.622, p < 0.0001), but this was not the case for UEtG and self-reported ethanol intake during the previous 7 days., Conclusion: (1) HEtG identified 20 cases of daily ethanol intake of more than 20 g, that would have been missed by the sole use of question 1 (frequency) and 2 (quantity) of the AUDIT. (2) Using the total score of the AUDIT, HEtG confirmed 10 more cases positive for alcohol intake. (3) Episodic heavy drinking is with 22.5% more frequent than in general population, and (4) of the 14 participants who reported no alcohol intake during the previous 7 days, 4 were UEtG positive. Improved detection of alcohol consumption, which is hazardous or harmful in the context of HCV and opiate dependence, would allow for earlier intervention in this population which is at particular risk of liver disease and fatal respiratory-depressed overdose. The combined use of self-reports and direct ethanol metabolites seems promising.

The epsilon4 allele of the apolipoprotein E gene (APOE4) has been consistently associated with a greater risk of Alzheimer's disease (AD) as well as an earlier onset of AD. It is possible that APOE4 may also play a role in the etiology of other neurodegenerative disorders, such as Parkinson's disease (PD). APOE genotype, age of onset, disease duration, smoking history, and dementia status were collected for families with PD, yielding 324 Caucasian families with complete information. Logistic regression employing one individual per family and including age of onset and disease duration as covariates demonstrated a significantly increased risk of dementia for those individuals having inherited at least one epsilon4 allele (OR=3.37; P=0.002). Survival analyses also demonstrated a significantly earlier age of onset for those subjects with at least one epsilon4 allele (59.7 years) as compared with those homozygous for the more common epsilon3 allele (62.4 years; P=0.009). Thus, consistent with previous studies, we find evidence that the presence of an epsilon4 allele results in significantly earlier onset of PD and a greater likelihood of dementia. It appears the similarities between PD and AD may be due to an overlap in the diseases' genetic etiology., (Copyright (c) 2005 Movement Disorder Society.)

Parkinson's disease (PD) is a common neurodegenerative disorder in humans with wide variability in the age of disease onset. Although the disease has been thought previously to occur sporadically in most patients, there is increasing evidence of a genetic contribution to the disorder. Recently, a polymorphic variant within intron 6 of the Nurr1 gene was reported to be associated with sporadic and familial PD. In an effort to identify susceptibility genes for PD, we have collected 783 PD patients from 372 families and 397 healthy controls from 217 families. PD patients and healthy controls were genotyped for the intron 6 insertion polymorphism by BseRI restriction endonuclease digestion. No significant difference in either homozygosity or heterozygosity for the 7048G7049 (IVS6 1361 +16insG) polymorphism was detected in the PD patient cohort as compared with the panel of healthy controls. Moreover, direct sequencing of exon 1 of the Nurr1 gene in PD patients failed to detect either of the two recently reported Nurr1 mutations identified in a small subset of a PD patient cohort. Taken together, these data suggest that genetic alteration at the Nurr1 locus is not a significant risk factor for the development of Parkinson's disease in our large sample of familial PD patients., (Copyright 2004 Movement Disorder Society)

Parkinson disease (PD) is the second most common neurodegenerative disorder. We studied 754 affected individuals, comprising 425 sibling pairs, to identify PD susceptibility genes. Screening of the parkin gene was performed in a subset of the sample having earlier age of PD onset or a positive LOD score with a marker in the parkin gene. All subjects were evaluated using a rigorous neurological assessment. Two diagnostic models were considered for genome-wide, non-parametric linkage analyses. Model I included only those individuals with a more stringent diagnosis of verified PD (216 sibling pairs) and resulted in a maximum LOD score of 3.4 on chromosome 2. Model II included all affected individuals (425 sibling pairs) and yielded a LOD score of 3.1 on the X chromosome. Our large sample was then employed to test for gene-by-gene (epistatic) interactions. A genome screen using the 23 families with PD patients having a mutation in only one allele of the parkin gene detected evidence of linkage to chromosome 10 (LOD=2.3). The 85 families with a very strong family history of PD were employed in a genome screen and, in addition to strong evidence of linkage to chromosome 2 (LOD=4.9), also produced a LOD of 2.4 on chromosome 14. A genome screen performed in the 277 families without a strong family history of PD detected linkage to chromosomes 10 (LOD=2.4) and X (LOD=3.2). These findings demonstrate consistent evidence of linkage to chromosomes 2 and X and also support the hypothesis that gene-by-gene interactions are important in PD susceptibility.

Parkinson disease (PD) is the second most common neurodegenerative disorder, surpassed in frequency only by Alzheimer disease. Elsewhere we have reported linkage to chromosome 2q in a sample of sibling pairs with PD. We have now expanded our sample to include 150 families meeting our strictest diagnostic definition of verified PD. To further delineate the chromosome 2q linkage, we have performed analyses using only those pedigrees with the strongest family history of PD. Linkage analyses in this subset of 65 pedigrees generated a LOD score of 5.1, which was obtained using an autosomal dominant model of disease transmission. This result strongly suggests that variation in a gene on chromosome 2q36-37 contributes to PD susceptibility.

Background: The vast majority of the parkin mutations previously identified have been found in individuals with juvenile or early onset PD. Previous screening of later onset PD cohorts has not identified substantial numbers of parkin mutations., Methods: Families with at least two siblings with PD were ascertained to identify genes contributing to PD susceptibility. Screening of the parkin gene, by both quantitative PCR and exon sequencing, was performed in those families with either early onset PD (age onset < or =50 years) or positive lod score with a marker in intron 7 of the parkin gene., Results: A total of 25 different mutations in the parkin gene were identified in 103 individuals from 47 families. Mutations were found in both parkin alleles in 41 of the individuals, whereas a single mutation in only one of the two parkin alleles was observed in 62 individuals. Thirty-five of the subjects (34%) with a parkin mutation had an age at onset of 60 years or above with 30 of these 35 (86%) having a detectable mutation on only one parkin allele. Few significant clinical differences were observed among the individuals with two, one, or no mutated copies of the parkin gene., Conclusion: Mutations in the parkin gene occur among individuals with PD with an older age at onset (> or =60 years) who have a positive family history of the disease. In addition, the clinical findings of parkin-positive individuals are remarkably similar to those without mutations.

Parkinson disease (PD) is a common neurodegenerative disorder characterized by bradykinesia, resting tremor, muscular rigidity, and postural instability, as well as by a clinically significant response to treatment with levodopa. Mutations in the alpha-synuclein gene have been found to result in autosomal dominant PD, and mutations in the parkin gene produce autosomal recessive juvenile-onset PD. We have studied 203 sibling pairs with PD who were evaluated by a rigorous neurological assessment based on (a) inclusion criteria consisting of clinical features highly associated with autopsy-confirmed PD and (b) exclusion criteria highly associated with other, non-PD pathological diagnoses. Families with positive LOD scores for a marker in an intron of the parkin gene were prioritized for parkin-gene testing, and mutations in the parkin gene were identified in 22 families. To reduce genetic heterogeneity, these families were not included in subsequent genome-screen analysis. Thus, a total of 160 multiplex families without evidence of a parkin mutation were used in multipoint nonparametric linkage analysis to identify PD-susceptibility genes. Two models of PD affection status were considered: model I included only those individuals with a more stringent diagnosis of verified PD (96 sibling pairs from 90 families), whereas model II included all examined individuals as affected, regardless of their final diagnostic classification (170 sibling pairs from 160 families). Under model I, the highest LOD scores were observed on chromosome X (LOD score 2.1) and on chromosome 2 (LOD score 1.9). Analyses performed with all available sibling pairs (model II) found even greater evidence of linkage to chromosome X (LOD score 2.7) and to chromosome 2 (LOD score 2.5). Evidence of linkage was also found to chromosomes 4, 5, and 13 (LOD scores >1.5). Our findings are consistent with those of other linkage studies that have reported linkage to chromosomes 5 and X.

Endocrine crises can occur in diabetes mellitus, in pituitary failure when there is a lack of ACTH, TSH or ADH secretion, in severe hyper- or hypothyroidism (thyroid storm and myxedema coma), severe hyper- or hypoparathyroidism (parathyroid crisis and tetany), in adrenal failure and in patients with pheochromocytoma or carcinoid tumors. Cushing's syndrome can be associated with psychotic crises. This review describes the most important clinical features and the basic diagnostic and therapeutic aspects of the non diabetic endocrine crises.