David Siemieniak

MATRIX-ASSISTED LASER DESORPTION/IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY AS A RAPID SCREENING METHOD TO DETECT MUTATIONS CAUSING TAY-SACHS DISEASE

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used as a rapid method for the detection of human genetic polymorphisms. In particular, the mutations in the human HEXA gene that cause the infantile Tay-Sachs disease have been studied using MALDI-MS to demonstrate the feasibility of this technique for use in clinical and diagnostic analysis. The protocols involved in this approach include, polymerase chain reaction for the amplification of the mutation site from buccal cell DNA, followed by restriction enzyme digestion of the amplified regions of the template cells. The products of amplification and digestion were studied using MALDI-MS. MALDI-MS experiments are shown to provide essentially the same information as obtained from gel electrophoresis but orders of magnitude faster.

Linkage of combined factors V and VIII deficiency to chromosome 18q by homozygosity mapping.

Combined Factors V and VIII deficiency is an autosomal recessive bleeding disorder identified in at least 58 families comprising a number of different ethnic groups. Affected patients present with a moderate bleeding tendency and have Factor V and Factor VIII levels in the range of 5-30% of normal. The highest frequency of the mutant gene is found in Jews of Sephardic and Middle Eastern origin living in Israel with an estimated disease frequency of 1:100,000. We sought to identify the gene responsible for combined Factors V and VIII deficiency using a positional cloning approach. Of 14 affected individuals from 8 unrelated Jewish families, 12 were the offspring of first-cousin marriages. After a genome-wide search using 241 highly polymorphic short tandem repeat (STR) markers, 13 of the 14 affected patients were homozygous for two closely linked 18q markers. Patients and all available family members were genotyped for 11 additional STRs spanning approximately 11 cM on the long arm of chromosome 18. Multipoint linkage analysis yielded a maximal log of the odds (LOD) score of 13.22. Haplotype analysis identified a number of recombinant individuals and established a minimum candidate interval of 2.5 cM for the gene responsible for combined Factors V and VIII deficiency. The product of this locus is likely to operate at a common step in the biosynthetic pathway for these two functionally and structurally homologous coagulation proteins. Identification of this gene should provide new insight into the biology of Factor V and Factor VIII production.

Linkage analysis identifies a locus for plasma von Willebrand factor undetected by genome-wide association

The plasma glycoprotein von Willebrand factor (VWF) exhibits fivefold antigen level variation across the normal human population determined by both genetic and environmental factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis, myocardial infarction, and stroke. To identify additional genetic determinants of VWF antigen levels and to minimize the impact of age and illness-related environmental factors, we performed genome-wide association analysis in two young and healthy cohorts (n = 1,152 and n = 2,310) and identified signals at ABO (P < 7.9E-139) and VWF (P < 5.5E-16), consistent with previous reports. Additionally, linkage analysis based on sibling structure within the cohorts, identified significant signals at chromosome 2q12–2p13 (LOD score 5.3) and at the ABO locus on chromosome 9q34 (LOD score 2.9) that explained 19.2% and 24.5% of the variance in VWF levels, respectively. Given its strong effect, the linkage region on chromosome 2 could harbor a potentially important determinant of bleeding and thrombosis risk. The absence of a chromosome 2 association signal in this or previous association studies suggests a causative gene harboring many genetic variants that are individually rare, but in aggregate common. These results raise the possibility that similar loci could explain a significant portion of the “missing heritability” for other complex genetic traits.

Genotyping of Apolipoprotein E by Matrix- assisted Laser Desorption/Ionization Time-of- flight Mass Spectrometry

The genotyping of the various isoforms of Apolipoprotein E (apo E) has been performed using matrix-assisted laser desorption/ionization (MALDI-MS). The polymerase chain reaction was used to amplify the specific apo E gene sequence followed by digestion with Cfo I (Clostridium formicoaceticum), for generating restriction fragments for rapid and accurate mass analysis. An exonuclease I digestion step was introduced to remove the unused primers after PCR, which can otherwise interfere in the mass spectral analysis. By replacing the gel electrophoresis detection step with MALDI-MS, restriction isotyping of the apo E gene was achieved. Genotyping of an unknown sample and obtained from an independent diagnostic laboratory demonstrated the validity of the MALDI-MS method for the routine analysis of apo E.

Absence of a Red Blood Cell Phenotype in Mice with Hematopoietic Deficiency of SEC23B

ABSTRACT Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease of ineffective erythropoiesis characterized by increased bi/multinucleated erythroid precursors in the bone marrow. CDAII results from mutations in SEC23B. The SEC23 protein is a core component of coat protein complex II-coated vesicles, which transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Though the genetic defect underlying CDAII has been identified, the pathophysiology of this disease remains unknown. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration, with this early mortality limiting evaluation of the adult hematopoietic compartment. We now report that mice with SEC23B deficiency restricted to the hematopoietic compartment survive normally and do not exhibit anemia or other CDAII characteristics. We also demonstrate that SEC23B-deficient hematopoietic stem cells (HSC) do not exhibit a disadvantage at reconstituting hematopoiesis when compared directly to wild-type HSC in a competitive repopulation assay. Secondary bone marrow transplants demonstrated continued equivalence of SEC23B-deficient and WT HSC in their hematopoietic reconstitution potential. The surprising discordance in phenotypes between SEC23B-deficient mice and humans may reflect an evolutionary shift in SEC23 paralog function and/or expression, or a change in a specific COPII cargo critical for erythropoiesis.

Analysis of Informed Consent Document Utilization in a Minimal-Risk Genetic Study

BACKGROUND The signed informed consent document certifies that the process of informed consent has taken place and provides research participants with comprehensive information about their role in the study. Despite efforts to optimize the informed consent document, only limited data are available about the actual use of consent documents by participants in biomedical research. OBJECTIVE To examine the use of online consent documents in a minimal-risk genetic study. DESIGN Prospective sibling cohort enrolled as part of a genetic study of hematologic and common human traits. SETTING University of Michigan Campus, Ann Arbor, Michigan. PARTICIPANTS Volunteer sample of healthy persons with 1 or more eligible siblings aged 14 to 35 years. Enrollment was through targeted e-mail to student lists. A total of 1209 persons completed the study. MEASUREMENTS Time taken by participants to review a 2833-word online consent document before indicating consent and identification of a masked hyperlink near the end of the document. RESULTS The minimum predicted reading time was 566 seconds. The median time to consent was 53 seconds. A total of 23% of participants consented within 10 seconds, and 93% of participants consented in less than the minimum predicted reading time. A total of 2.5% of participants identified the masked hyperlink. LIMITATION The online consent process was not observed directly by study investigators, and some participants may have viewed the consent document more than once. CONCLUSION Few research participants thoroughly read the consent document before agreeing to participate in this genetic study. These data suggest that current informed consent documents, particularly for low-risk studies, may no longer serve the intended purpose of protecting human participants, and the role of these documents should be reassessed. PRIMARY FUNDING SOURCE National Institutes of Health.

Genome-wide studies of von Willebrand factor propeptide identify loci contributing to variation in propeptide levels and von Willebrand factor clearance.

Essentials Variants at ABO, von Willebrand Factor (VWF) and 2q12 contribute to the variation in plasma in VWF. We performed a genome‐wide association study of plasma VWF propeptide in 3,238 individuals. ABO, VWF and 2q12 loci had weak or no association or linkage with plasma VWFpp levels. VWF associated variants at ABO, VWF and 2q12 loci primarily affect VWF clearance rates.

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

Significance Here we report a method to rapidly examine the effect of nearly all possible single amino acid substitutions within a substrate fragment of the coagulation protein von Willebrand factor (VWF) on the efficiency of cleavage by its cognate protease, ADAMTS13. A substrate phage display library was generated containing ∼3.5 × 107 independent clones and uncleaved phages collected at multiple reaction time points after reaction with ADAMTS13. Analysis of these phages by high-throughput sequencing facilitated simultaneous calculations of kcat/KM values for multiple substitutions at each position of this protein fragment, providing a comprehensive picture of the substrate recognition landscape for the interaction between ADAMTS13 and VWF. This approach should be broadly applicable to many other protease/substrate pairs. Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (kcat/KM) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1′ substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.

Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci

Significance Venous thromboembolism (VTE) is a common disease characterized by the formation of inappropriate blood clots. Inheritance of specific genetic variants, such as the Factor V Leiden polymorphism, increases VTE susceptibility. However, only ∼10% of people inheriting Factor V Leiden develop VTE, suggesting the involvement of other genes that are currently unknown. By inducing random genetic mutations into mice with a genetic predisposition to VTE, we identified two genomic regions that reduce VTE susceptibility. The first includes the gene for blood coagulation, Factor 3, and its role was confirmed by analyzing mice with an independent mutation in this gene. The second contains a mutation in the Actr2 gene. These findings identify critical genes for the regulation of blood-clotting risk. Factor V Leiden (F5L) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5L (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/−). F8 deficiency enhanced the survival of F5L/L Tfpi+/− mice, demonstrating that F5L/L Tfpi+/− lethality is genetically suppressible. ENU-mutagenized F5L/L males and F5L/+ Tfpi+/− females were crossed to generate 6,729 progeny, with 98 F5L/L Tfpi+/− offspring surviving until weaning. Sixteen lines, referred to as “modifier of Factor 5 Leiden (MF5L1–16),” exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3+/−) suppressed F5L/L Tfpi+/− lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5L/L Tfpi+/− lethality (P = 1.7 × 10−6), suggesting that Actr2p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2p.R258G. Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5L and also suggest a role for Actr2 in this process.

Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse

Although the Factor V Leiden (FVL) gene variant is the most prevalent genetic risk factor for venous thrombosis, only 10% of FVL carriers will experience such an event in their lifetime. To identify potential FVL modifier genes contributing to this incomplete penetrance, we took advantage of a perinatal synthetic lethal thrombosis phenotype in mice homozygous for FVL (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/-) to perform a sensitized dominant ENU mutagenesis screen. Linkage analysis conducted in the 3 largest pedigrees generated from the surviving F5L/L Tfpi+/- mice (‘rescues’) using ENU-induced coding variants as genetic markers was unsuccessful in identifying major suppressor loci. Whole exome sequencing was applied to DNA from 107 rescue mice to identify candidate genes enriched for ENU mutations. A total of 3,481 potentially deleterious candidate ENU variants were identified in 2,984 genes. After correcting for gene size and multiple testing, Arl6ip5 was identified as the most enriched gene, though not reaching genome-wide significance. Evaluation of CRISPR/Cas9 induced loss of function in the top 6 genes failed to demonstrate a clear rescue phenotype. However, a maternally inherited (not ENU-induced) de novo mutation (Plcb4R335Q) exhibited significant co-segregation with the rescue phenotype (p = 0.003) in the corresponding pedigree. Thrombosis suppression by heterozygous Plcb4 loss of function was confirmed through analysis of an independent, CRISPR/Cas9-induced Plcb4 mutation (p = 0.01).