A proactive genotype-to-patient-phenotype map for cystathionine beta-synthase

Sun, Song; Verby, Marta; Coté, Atina G.; Wu, Yingzhou; Fotiadou, Iosifina; Kitaygorodsky, Julia; Rine, Jasper; Ješina, Pavel; Kožich, Viktor; Roth, Frederick P.

doi:10.1101/473983

Cited by 18 publications

(54 citation statements)

References 52 publications

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“…In terms of overall constraint, the dataset closest to MSH2 is from the transcription factor PPARG, in which 21.5% of missense variants scored as having substantial probability of being casual for lipodystrophy 76 . Most other human genes subjected to full-length mutational scans thus far have shown substantially higher overall constraint: for instance, 40.5% of missense variants in the pharmacogene NUDT15 scored as damaging 62 , as were 39% of variants in the homocysteine metabolic factor CBS 77 . A recent activity-agnostic protein stability screen showed the fraction of missense variants with substantially destabilizing effects to range from 25.1-43.1% across three different genes 62,78 ; these could be taken as a lower bound given that an unknown fraction of true LOF variants may remain stable.…”

Section: Discussionmentioning

confidence: 99%

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Jia

Burugula

Chen

et al. 2020

Preprint

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Introduction 3The lack of functional evidence for the majority of missense variants limits their clinical interpretability, 4 and poses a key barrier to the broad utility of carrier screening. In Lynch Syndrome (LS), one of the most 5 highly prevalent cancer syndromes, nearly 90% of clinically observed missense variants are deemed 6 'variants of uncertain significance' (VUS). To systematically resolve their functional status, we performed 7 a massively parallel screen in human cells to identify loss-of-function missense variants in the key DNA 8 mismatch repair factor MSH2. The resulting sequence-function map is substantially complete, covering 9 94% of the 17,746 possible variants, and is highly concordant (≥96%) with existing functional data and 10 expert clinicians' interpretations. The large majority (89%) of missense variants were functionally neutral, 11 perhaps unexpectedly in view of its evolutionary conservation. These data provide ready-to-use 12 functional evidence to resolve the ~1,300 extant missense VUSs in MSH2, and may facilitate the 13 prospective classification of newly discovered variants in the clinic. 14 15 Main text 16 17 Lynch Syndrome (LS, OMIM:120435), the first discovered familial cancer syndrome 1 , confers 18 predisposition to colorectal and endometrial cancers due to the loss of DNA mismatch repair (MMR) and 19 resulting mutagenic burden. Most affected individuals inherit a single loss-of-function variant in one of 20 four MMR factors (MSH2, MLH1, 21 MSH6, PMS2), followed by a somatic 22 'second hit' inactivating the remaining 23 functional copy. Due to high 24 population prevalence (≥1:300) 2,3 , 25 clear genetic etiology, and potential 26 for prevention through intensified 27 surveillance, MMR gene variants are 28 considered clinically actionable 4 . 29 30 Missense changes together comprise 31 20-30% of LS variants 5 , and are 32 particularly challenging to interpret: 33 their functional impacts may range 34 from minimal to profound, most are 35 individually rare, and they exhibit 36 incomplete penetrance 6 . 37 Consequently, the overwhelming 38 majority of LS gene missense variants 39 listed in ClinVar (4,762/5,473, 87.0%) 40 are deemed 'variants of uncertain 41 significance', or VUS 42( Supplementary Fig.1), and cannot 43 be used to guide diagnosis. 45To address these challenges, we 46 performed a deep mutational scan 7 to 47 systematically measure the functional 48 impact of missense variants in the 49 major Lynch Syndrome gene MSH2. 50We established a human cell system 51 to model MSH2 variant function using 52 the mismatch repair proficient 8 cell 53 line HAP1 (Fig. 1a,c). First, to disrupt MMR, we derived clonal MSH2 knockout cells bearing a 19-bp frameshift deletion in MSH2 exon 6, and 55 verified that it lacked detectable MSH2 protein expression ( Supplementary Fig. 2). To restore MMR, we 56 stably reintroduced into cells either wild-type MSH2 cDNA (KO+WT) or a pathogenic founder allele 57 (KO+A636P) on inducible lentiviral constructs. After inducing expression with doxycycl...

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Section: Discussionmentioning

confidence: 99%

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Jia

Burugula

Chen

et al. 2020

Preprint

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“…9 It is increasingly clear that testing variant functions ''reactively'' (only after first observing the variant in a human) cannot keep pace. A more ''proactive'' approach, in which multiplexed assays of variant effect (MAVEs) are applied to systematically test variants in disease-associated genes, is emerging; such variants include missense variants not yet seen in any human, 10,11 e.g., for cystathionine beta-synthase (CBS) 12 and calmodulin. 10 Here, we examine the human gene MTHFR, encoding 5,10-methylenetetrahydrofolate reductase, a key enzyme in the one-carbon metabolism pathway that includes the essential folate and methionine cycles.…”

Section: Introductionmentioning

confidence: 99%

Shifting landscapes of human MTHFR missense-variant effects

Kishore

Sun

Maaieh

et al. 2021

The American Journal of Human Genetics

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Most rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment-and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency.

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“…These humanized strains can now serve as cellular reagents to study complex human cytoskeletal processes in a simplified eukaryotic context, allowing functional roles of distinct family members to be assayed individually. Screening allelic variants and mutational libraries using these strains might enable the rapid identification of disease variants in a high-throughput manner ( Kachroo et al 2015 ; Hamza et al 2015 ; Sun et al 2016 , 2018 ; Yang et al 2017 ; Marzo et al 2019 ), paving the way for a better understanding of the genetic and molecular basis of cytoskeletal disorders.…”

Section: Discussionmentioning

confidence: 99%

Systematic Humanization of the Yeast Cytoskeleton Discerns Functionally Replaceable from Divergent Human Genes

et al. 2020

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Many gene families have been expanded by gene duplications along the human lineage, relative to ancestral opisthokonts, but the extent to which the duplicated genes function similarly is understudied. Here, we focused on structural cytoskeletal genes involved in critical cellular processes including chromosome segregation, macromolecular transport, and cell shape maintenance. To determine functional redundancy and divergence of duplicated human genes, we systematically humanized the yeast actin, myosin, tubulin, and septin genes, testing ~81% of human cytoskeletal genes across 7 gene families for their ability to complement a growth defect induced by inactivation or deletion of the corresponding yeast ortholog. In 5 of 7 families-all but α-tubulin and light myosin, we found at least one human gene capable of complementing loss of the yeast gene. Despite rescuing growth defects, we observed differential abilities of human genes to rescue cell morphology, meiosis, and mating defects. By comparing phenotypes of humanized strains with deletion phenotypes of their interaction partners, we identify instances of human genes in the actin and septin families capable of carrying out essential functions, but apparently failing to fully complement the cytoskeletal roles of their yeast orthologs, thus leading to abnormal cell morphologies. Overall, we show that duplicated human cytoskeletal genes appear to have diverged such that only a few human genes within each family are capable of replacing the essential roles of their yeast orthologs. The resulting yeast strains with humanized cytoskeletal components now provide surrogate platforms to characterize human genes in simplified eukaryotic contexts.

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A proactive genotype-to-patient-phenotype map for cystathionine beta-synthase

Cited by 18 publications

References 52 publications

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Shifting landscapes of human MTHFR missense-variant effects

Systematic Humanization of the Yeast Cytoskeleton Discerns Functionally Replaceable from Divergent Human Genes

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