Bayesian model for accurate MARSALA (mutated allele revealed by sequencing with aneuploidy and linkage analyses).

PURPOSE: This study is aimed at increasing the accuracy of preimplantation genetic test for monogenic defects (PGT-M). METHODS: We applied Bayesian statistics to optimize data analyses of the mutated allele revealed by sequencing with aneuploidy and linkage analyses (MARSALA) method for PGT-M. In doing so, we developed a Bayesian algorithm for linkage analyses incorporating PCR SNV detection with genome sequencing around the known mutation sites in order to determine quantitatively the probabilities of having the disease-carrying alleles from parents with monogenic diseases. Both recombination events and sequencing errors were taken into account in calculating the probability. RESULTS: Data of 28 in vitro fertilized embryos from three couples were retrieved from two published research articles by Yan et al. (Proc Natl Acad Sci. 112:15964-9, 2015) and Wilton et al. (Hum Reprod. 24:1221-8, 2009). We found the embryos deemed "normal" and selected for transfer in the previous publications were actually different in error probability of 10-4-4%. Notably, our Bayesian model reduced the error probability to 10-6-10-4%. Furthermore, a proband sample is no longer required by our new method, given a minimum of four embryos or sperm cells. CONCLUSION: The error probability of PGT-M can be significantly reduced by using the Bayesian statistics approach, increasing the accuracy of selecting healthy embryos for transfer with or without a proband sample.

Haplotyping by linked-read sequencing (HLRS) of the genetic disease carriers for preimplantation genetic testing without a proband or relatives.

BACKGROUND: In order to mitigate the risk of allele dropout (ADO) and ensure the accuracy of preimplantation genetic testing for monogenic disease (PGT-M), it is necessary to construct parental haplotypes. Typically, haplotype resolution is obtained by genotyping multiple polymorphic markers in both parents and a proband or a relative. Sometimes, single sperm typing, or tests on the polar bodies may also be useful. Nevertheless, this process is time-consuming. At present, there was no simple linkage analysis strategy for patients without affected relatives. METHOD: To solve this problem, we established a haplotyping by linked-read sequencing (HLRS) method without the requirement for additional relatives. First, the haplotype of the genetic disease carriers in the family was constructed by linked-read sequencing, and then the informative single nucleotide polymorphisms (SNPs) in upstream and downstream mutation region were selected to construct the embryo haplotype and to determine whether the embryo was carrying the mutation. Two families were selected to validate this method; one with alpha thalassemia and the other with NDP gene disorder. RESULTS: The haplotyping by linked-read sequencing (HLRS) method was successfully applied to construct parental haplotypes without recruiting additional family members; the method was also validated for PGT-M. The mutation carriers in these families were sequenced by linked-read sequencing, and their haplotypes were successfully phased. Adjacent SNPs of the mutation gene were identified. The informative SNPs were chosen for linkage analyses to identify the carrier embryos. For the alpha thalassemia family, a normal blastocyst was transferred to the uterus and the accuracy of PGT-M was confirmed by amniocentesis at 16 weeks of gestation. CONCLUSIONS: Our results suggest that HLRS can be applied for PGT-M of monogenic disorders or de novo mutations where the mutations haplotype cannot be determined due to absence of affected relatives.

Linkage, whole genome sequence, and biological data implicate variants in RAB10 in Alzheimer's disease resilience.

BACKGROUND: While age and the APOE ε4 allele are major risk factors for Alzheimer's disease (AD), a small percentage of individuals with these risk factors exhibit AD resilience by living well beyond 75 years of age without any clinical symptoms of cognitive decline. METHODS: We used over 200 "AD resilient" individuals and an innovative, pedigree-based approach to identify genetic variants that segregate with AD resilience. First, we performed linkage analyses in pedigrees with resilient individuals and a statistical excess of AD deaths. Second, we used whole genome sequences to identify candidate SNPs in significant linkage regions. Third, we replicated SNPs from the linkage peaks that reduced risk for AD in an independent dataset and in a gene-based test. Finally, we experimentally characterized replicated SNPs. RESULTS: Rs142787485 in RAB10 confers significant protection against AD (p value = 0.0184, odds ratio = 0.5853). Moreover, we replicated this association in an independent series of unrelated individuals (p value = 0.028, odds ratio = 0.69) and used a gene-based test to confirm a role for RAB10 variants in modifying AD risk (p value = 0.002). Experimentally, we demonstrated that knockdown of RAB10 resulted in a significant decrease in Aß42 (p value = 0.0003) and in the Aß42/Aß40 ratio (p value = 0.0001) in neuroblastoma cells. We also found that RAB10 expression is significantly elevated in human AD brains (p value = 0.04). CONCLUSIONS: Our results suggest that RAB10 could be a promising therapeutic target for AD prevention. In addition, our gene discovery approach can be expanded and adapted to other phenotypes, thus serving as a model for future efforts to identify rare variants for AD and other complex human diseases.

Eigenvector metabolite analysis reveals dietary effects on the association among metabolite correlation patterns, gene expression, and phenotypes.

INTRODUCTION: 'Multi-omics' datasets obtained from an organism of interest reared under different environmental treatments are increasingly common. Identifying the links among metabolites and transcripts can help to elucidate our understanding of the impact of environment at different levels within the organism. However, many methods for characterizing physiological connections cannot address unidentified metabolites. OBJECTIVES: Here, we use Eigenvector Metabolite Analysis (EvMA) to examine links between metabolomic, transcriptomic, and phenotypic variation data and to assess the impact of environmental factors on these associations. Unlike other methods, EvMA can be used to analyze datasets that include unidentified metabolites and unannotated transcripts. METHODS: To demonstrate the utility of EvMA, we analyzed metabolomic, transcriptomic, and phenotypic datasets produced from 20 Drosophila melanogaster genotypes reared on four dietary treatments. We used a hierarchical distance-based method to cluster the metabolites. The links between metabolite clusters, gene expression, and overt phenotypes were characterized using the eigenmetabolite (first principal component) of each cluster. RESULTS: EvMA recovered chemically related groups of metabolites within the clusters. Using the eigenmetabolite, we identified genes and phenotypes that significantly correlated with each cluster. EvMA identifies new connections between the phenotypes, metabolites, and gene transcripts. Conclusion EvMA provides a simple method to identify correlations between metabolites, gene expression, and phenotypes, which can allow us to partition multivariate datasets into meaningful biological modules and identify under-studied metabolites and unannotated gene transcripts that may be central to important biological processes. This can be used to inform our understanding of the effect of environmental mechanisms underlying physiological states of interest.

Clinical applications of MARSALA for preimplantation genetic diagnosis of spinal muscular atrophy.

Conventional PCR methods combined with linkage analysis based on short tandem repeats (STRs) or Karyomapping with single nucleotide polymorphism (SNP) arrays, have been applied to preimplantation genetic diagnosis (PGD) for spinal muscular atrophy (SMA), an autosome recessive disorder. However, it has limitations in SMA diagnosis by Karyomapping, and these methods are unable to distinguish wild-type embryos with carriers effectively. Mutated allele revealed by sequencing with aneuploidy and linkage analyses (MARSALA) is a new method allowing embryo selection by a one-step next-generation sequencing (NGS) procedure, which has been applied in PGD for both autosome dominant and X-linked diseases in our group previously. In this study, we carried out PGD based on MARSALA for two carrier families with SMA affected children. As a result, one of the couples has given birth to a healthy baby free of mutations in SMA-causing gene. It is the first time that MARSALA was applied to PGD for SMA, and we can distinguish the embryos with heterozygous deletion (carriers) from the wild-type (normal) ones accurately through this NGS-based method. In addition, direct mutation detection allows us to identify the affected embryos (homozygous deletion), which can be regarded as probands for linkage analysis, in case that the affected family member is absent. In the future, the NGS-based MARSALA method is expected to be used in PGD for all monogenetic disorders with known pathogenic gene mutation.

The Genetics of IgA Nephropathy: An Overview from China.

BACKGROUND: IgA nephropathy (IgAN) is the most common type of primary glomerulonephritis worldwide. Highly variable data for disease prevalence and reports of familial clustering suggest the involvement of genetic factors in IgAN. As China is an area with a high prevalence of IgAN, Chinese scholars have made a considerable effort to reveal the underlying genetic architecture of IgAN. SUMMARY: In this review, we summarize recent achievements in the genetic studies of IgAN, focusing mainly on studies undertaken in China. Early association studies followed a population-based design and focused on a single variant or single gene. Subsequently, family-based designs and genetic interactions applied by Chinese scholars revealed an association of variants in MEGSIN and glycosyltransferase genes with IgAN. Recently, genome-wide association studies (GWAS) have been used to identify multiple susceptibility loci for IgAN, and they have, for the most part, been validated in Chinese populations. KEY MESSAGE: More efforts should be made to explore the underlying genetic mechanisms of GWAS-identified variants. In future studies in IgAN, the application of a systems genetics approach would be helpful and productive. FACTS FROM EAST AND WEST: The reported prevalence of IgAN is higher in Asia than in Europe and North America. However, differences in use of biopsy for the diagnosis of IgAN should be taken into account in analyzing data from both East and West. In Europe, IgAN affects men more frequently than women; this is not the case in Asia. Familial IgAN has been more frequently reported in Europe than in Asia. Within Europe, familial IgAN is more evident in southern than in northern populations. Changes in the pattern of serum IgA1 O-glycosylation is a common finding in IgAN patients in the East and West. SNPs within the gene coding for the enzyme C1GALT1 have been reported in Chinese and European patients. However, there is no evidence for a role of gene polymorphism of the C1GALT1 chaperone cosmc in Europeans. Genetic variants in the HLA gene family have been observed in populations from the East and West. Associations between IgAN and variants of the TAP1/PSMB and DEFA genes were observed in Asian but not in Western patients. Association with the angiotensin-converting enzyme gene was seen only in Asian patients.