Integrating sequence and array data to create an improved 1000 Genomes Project haplotype reference panel

A major use of the 1000 Genomes Project (1000GP) data is genotype imputation in genome-wide association studies (GWAS). Here we develop a method to estimate haplotypes from low-coverage sequencing data that can take advantage of single-nucleotide polymorphism (SNP) microarray genotypes on the same samples. First the SNP array data are phased to build a backbone (or 'scaffold') of haplotypes across each chromosome. We then phase the sequence data 'onto' this haplotype scaffold. This approach can take advantage of relatedness between sequenced and non-sequenced samples to improve accuracy. We use this method to create a new 1000GP haplotype reference set for use by the human genetic community. Using a set of validation genotypes at SNP and bi-allelic indels we show that these haplotypes have lower genotype discordance and improved imputation performance into downstream GWAS samples, especially at low-frequency variants. © 2014 Macmillan Publishers Limited. All rights reserved

Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder

Methylmalonic aciduria and homocystinuria, cblC type, is the most common inborn error of cellular vitamin B12 metabolism. We previously showed that the protein carrying the mutation responsible for late-onset cblC (MMACHC-R161Q), treatable with high dose OHCbl, is able to bind OHCbl with wild-type affinity, leaving undetermined the disease mechanism involved [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. To assess whether the mutation renders the protein unstable, we investigated the thermostability of the wild-type and mutant MMACHC proteins, either unbound or bound to different cobalamins (Cbl), using differential scanning fluorimetry. We found that MMACHC-wt and MMACHC-R161Q are both very thermolabile proteins in their apo forms, with melting temperatures (Tm) of 39.3 ± 1.0 and 37.1 ± 0.7 °C, respectively; a difference confirmed by unfolding of MMACHC-R161Q but not MMACHC-wt by isothermal denaturation at 35 °C over 120 min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (ΔTm max = 8 °C, half-maximal effective ligand concentration, AC50 = 3 μM). We surveyed the effect of different cobalamins on the stabilization of the wild-type protein and found that AdoCbl was the most stabilizing, exerting a maximum increase in Tm of ∼16 °C, followed by MeCbl at ∼13 °C, each evaluated at 50 μM cofactor. The other cobalamins stabilized in the order (CN)2Cbi > OHCbl > CNCbl. Interestingly, the AC50’s for AdoCbl, MeCbl, (CN)2Cbi and OHCbl were similar and ranged from 1–3 μM, which compares well with the Kd of 6 μM for OHCbl [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. Unlike MMACHC-wt, the mutant protein MMACHC-R161Q is only moderately stabilized by OHCbl (ΔTm max = 4 °C). The dose–response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC50 of 7 μM. MMACHC-R161Q showed the same order of stabilization as MMACHC-wt, but each cobalamin stabilized this mutant protein less than its wild-type counterpart. Additionally, MMACHC-R161Q had a higher AC50 for each cobalamin form compared to MMACHC-wt. Finally, we show that MMACHC-R161Q is able to support the base-off transition for AdoCbl and CNCbl, indicating this mutant is not blocked in that respect. Taken together, our results suggest that protein stability, as well as propensity for ligand-induced stabilization, contributes to the disease mechanism in late-onset cblC disorder. Our results underscore the importance of cofactor stabilization of MMACHC and suggest that even small increases in the concentration of cobalamin complexed with MMACHC may have therapeutic benefit in children with the late-onset, vitamin responsive cblC disease

Identification of candidate active site residues in lysosomal β- hexosaminidase A

The β-hexosaminidases (Hex) catalyze the cleavage of terminal amino sugars on a broad spectrum of glycoconjugates. The major Hex isozymes in humans, Hex A, a heterodimer of α and β subunits (αβ), and Hex B, a homodimer of β subunits (ββ), have different substrate specificities. The β subunit (HEXB gene product), hydrolyzes neutral substrates. The α subunit (HEXA gene product), hydrolyzes both neutral and charged substrates. Only Hex A is able to hydrolyze the most important natural substrate, the acidic glycolipid GM2 ganglioside. Mutations in the HEXA gene cause Tay-Sachs disease (TSD), a GM2 ganglioside storage disorder. We investigated the role of putative active site residues Asp-α258, Glu-α307, Glu-α323, and Glu- α462 in the α subunit of Hex A. A mutation at codon 258 which we described was associated with the TSD B1 phenotype, characterized by the presence of normal amounts of mature but catalytically inactive enzyme. TSD-B1 mutations are believed to involve substitutions of residues at the enzyme active site. Glu-α307, Glu-α323, and Glu-α462 were predicted to be active site residues by homology studies and hydrophobic cluster analysis. We used site-directed mutagenesis and expression in a novel transformed human fetal TSD neuroglial (TSD-NG) cell line (with very low levels of endogenous Hex A activity), to study the effects of mutation at candidate active site residues. Mutant HEXA cDNAs carrying conservative or isofunctional substitutions at these positions were expressed in TSD-NG cells. αE323D, αE462D, and αD258N cDNAs produced normally processed peptide chains with drastically reduced activity toward the α subunit-specific substrate 4MUGS. The αE307D cDNA produced a precursor peptide with significant catalytic activity. Kinetic analysis of enzymes carrying mutations at Glu-α323 and Asp-α258 (reported earlier by Bayleran, J., Hechtman, P., Kolodny, E., and Kaback, M. (1987) Am. J. Hum. Genet. 41,532-548) indicated no significant change in substrate binding properties. Our data, viewed in the context of homology studies and modeling, and studies with suicide substrates, suggest that Glu-α323 and Asp. α258 are active site residues and that Glu-α323 is involved in catalysis