A novel mutation of the SLC25A13 gene in a Chinese patient with citrin deficiency detected by target next-generation sequencing

Type II citrullinaemia, also known as citrin deficiency, is an autosomal recessive metabolic disorder, which is caused by pathogenic mutations in the SLC25A13 gene on chromosome 7q21.3. One of the clinical manifestations of type II citrullinaemia is neonatal intrahepatic cholestatic hepatitis caused by citrin deficiency (NICCD, OMIM# 605814). In this study, a 5-month-old female Chinese neonate diagnosed with type II citrullinaemia was examined. The diagnosis was based on biochemical and clinical findings, including organic acid profiling using a gas chromatography mass spectrometry (GC/MS), and the patient's parents were unaffected. Approximately 14 kb of the exon sequences of the SLC25A13 and two relative genes (ASS1 and FAH) from the proband and 100 case-unrelated controls were captured by array-based capture method followed by high-throughput next-generation sequencing. Two single-nucleotide mutations were detected in the proband, including the previous reported c.1177+1G>A mutation and a novel c.754G>A mutation in the SLC25A13 gene. Sanger sequence results showed that the patient was a compound heterozygote for the two mutations. The novel mutation (c.754G>A), which is predicted to affect the normal structure and function of citrin, is a candidate pathogenic mutation. Target sequence capture combined with high-throughput next-generation sequencing technologies is proven to be an effective method for molecular genetic testing of type II citrullinaemia.     

Mutation of SLC35D3 Causes Metabolic Syndrome by Impairing Dopamine Signaling in Striatal D1 Neurons

Obesity is one of the largest health problems facing the world today. Although twin and family studies suggest about two-thirds of obesity is caused by genetic factors, only a small fraction of this variance has been unraveled. There are still large numbers of genes to be identified that cause variations in body fatness and the associated diseases encompassed in the metabolic syndrome (MetS). A locus near a sequence tagged site (STS) marker D6S1009 has been linked to obesity or body mass index (BMI). However, its genetic entity is unknown. D6S1009 is located in the intergenic region between SLC35D3 and NHEG1. Here we report that the ros mutant mice harboring a recessive mutation in the Slc35d3 gene show obesity and MetS and reduced membrane dopamine receptor D1 (D1R) with impaired dopamine signaling in striatal neurons. SLC35D3 is localized to both endoplasmic reticulum (ER) and early endosomes and interacts with D1R. In ros striatal D1 neurons, lack of SLC35D3 causes the accumulation of D1R on the ER to impair its ER exit. The MetS phenotype is reversible by the administration of D1R agonist to the ros mutant. In addition, we identified two mutations in the SLC35D3 gene in patients with MetS, which alter the subcellular localization of SLC35D3. Our results suggest that the SLC35D3 gene, close to the D6S1009 locus, is a candidate gene for MetS, which is involved in metabolic control in the central nervous system by regulating dopamine signaling.     

SNP-based pool genotyping and haplotype analysis accelerate fine-mapping of the wheat genomic region containing stripe rust resistance gene <Emphasis Type="Italic">Yr26</Emphasis>

Key message

NGS-assisted super pooling emerging as powerful tool to accelerate gene mapping and haplotype association analysis within target region uncovering specific linkage SNPs or alleles for marker-assisted gene pyramiding.

Abstract

Conventional gene mapping methods to identify genes associated with important agronomic traits require significant amounts of financial support and time. Here, a single nucleotide polymorphism (SNP)-based mapping approach, RNA-Seq and SNP array assisted super pooling analysis, was used for rapid mining of a candidate genomic region for stripe rust resistance gene Yr26 that has been widely used in wheat breeding programs in China. Large DNA and RNA super-pools were genotyped by Wheat SNP Array and sequenced by Illumina HiSeq, respectively. Hundreds of thousands of SNPs were identified and then filtered by multiple filtering criteria. Among selected SNPs, over 900 were found within an overlapping interval of less than 30 Mb as the Yr26 candidate genomic region in the centromeric region of chromosome arm 1BL. The 235 chromosome-specific SNPs were converted into KASP assays to validate the Yr26 interval in different genetic populations. Using a high-resolution mapping population (>?30,000 gametes), we confined Yr26 to a 0.003-cM interval. The Yr26 target region was anchored to the common wheat IWGSC RefSeq v1.0 and wild emmer WEWSeq v.1.0 sequences, from which 488 and 454 kb fragments were obtained. Several candidate genes were identified in the target genomic region, but there was no typical resistance gene in either genome region. Haplotype analysis identified specific SNPs linked to Yr26 and developed robust and breeder-friendly KASP markers. This integration strategy can be applied to accelerate generating many markers closely linked to target genes/QTL for a trait of interest in wheat and other polyploid species.

     

Two Genomic Loci Control Three Eye Colors in the Domestic Pigeon (Columba livia)

The iris of the eye shows striking color variation across vertebrate species, and may play important roles in crypsis and communication. The domestic pigeon (Columba livia) has three common iris colors, orange, pearl (white), and bull (dark brown), segregating in a single species, thereby providing a unique opportunity to identify the genetic basis of iris coloration. We used comparative genomics and genetic mapping in laboratory crosses to identify two candidate genes that control variation in iris color in domestic pigeons. We identified a nonsense mutation in the solute carrier SLC2A11B that is shared among all pigeons with pearl eye color, and a locus associated with bull eye color that includes EDNRB2, a gene involved in neural crest migration and pigment development. However, bull eye is likely controlled by a heterogeneous collection of alleles across pigeon breeds. We also found that the EDNRB2 region is associated with regionalized plumage depigmentation (piebalding). Our study identifies two candidate genes for eye colors variation, and establishes a genetic link between iris and plumage color, two traits that vary widely in the evolution of birds and other vertebrates.     

Mapping of the chicken cleft primary palate mutation on chromosome 11 and sequencing of the 4.9 Mb linked region

An embryonic lethal mutation in chicken named cleft primary palate (cpp) is inherited in an autosomal recessive mode and results in a severely truncated upper beak. In this study, genotyping and sequencing techniques were employed to advance our genetic and genomic knowledge of the mutation’s chromosomal location, candidate region and possible causative element using a congenic inbred line. Herein, the candidate region for the cpp developmental mutation was established as a ca. 5.1 Mb region of chicken chromosome 11 (GGA 11) through the use of a 600K Affymetrix SNP array. The SNPs identified from this array linked to cpp were used to genotype individuals from the congenic inbred line over several generations and thereby fine-map the causative region resulting in an approximately 200 kb size reduction. This candidate region (4.9 Mb) was sequenced via capture array in a cohort of 24 individuals, including carriers, mutants and their wild type (wt) siblings. Interestingly, the GGA 11 region for cpp encompasses the predicted centromere location and is thus unlikely to be highly disrupted by further recombination. Here we report on the variation unique to the cpp mutation, i.e. single-nucleotide variants and insertions or deletions. Although the candidate region contains several genes of interest with regard to the cpp phenotype, only one cpp-linked variant was predicted to have a significant physiological effect by causing a frameshift mutation in ESRP2, which has a role in tissue-specific splicing during development.     

Genomic Organization and Complete Nucleotide Sequence of the HumanPWP2Gene on Chromosome 21

The humanPWP2gene is the human homologue of the yeast periodic tryptophan protein 2 (PWP2) gene and is a member of the gene family that contains tryptophan-aspartate (WD) repeats. Genomic sequencing revealed that the humanPWP2gene consists of 21 exons spanning approximately 24 kb and locates just between the two genes EHOC-1 and KNP-I and distal to aNotI site of LJ104 (D21S1460) on chromosome 21q22.3. Analysis of the 5′-flanking DNA sequence revealed that the upstream region of thePWP2gene is associated with a CpG island containing theNotI site of LJ104. SincePWP2is considered to be a candidate for genetic disorders mapped in the 21q22.3 region, the information including nucleotide sequence and genomic organization of thePWP2gene should be invaluable for the mutation analysis of the corresponding genetic disorders.     

A Multi-Breed Genome-Wide Association Analysis for Canine Hypothyroidism Identifies a Shared Major Risk Locus on CFA12

Hypothyroidism is a complex clinical condition found in both humans and dogs, thought to be caused by a combination of genetic and environmental factors. In this study we present a multi-breed analysis of predisposing genetic risk factors for hypothyroidism in dogs using three high-risk breeds—the Gordon Setter, Hovawart and the Rhodesian Ridgeback. Using a genome-wide association approach and meta-analysis, we identified a major hypothyroidism risk locus shared by these breeds on chromosome 12 (p = 2.1x10^-11). Further characterisation of the candidate region revealed a shared ~167 kb risk haplotype (4,915,018–5,081,823 bp), tagged by two SNPs in almost complete linkage disequilibrium. This breed-shared risk haplotype includes three genes (LHFPL5, SRPK1 and SLC26A8) and does not extend to the dog leukocyte antigen (DLA) class II gene cluster located in the vicinity. These three genes have not been identified as candidate genes for hypothyroid disease previously, but have functions that could potentially contribute to the development of the disease. Our results implicate the potential involvement of novel genes and pathways for the development of canine hypothyroidism, raising new possibilities for screening, breeding programmes and treatments in dogs. This study may also contribute to our understanding of the genetic etiology of human hypothyroid disease, which is one of the most common endocrine disorders in humans.     

Construction of YAC Contigs at Human Chromosome 11q22.3-q23.1 Region Covering the Ataxia Telangiectasia Locus

Ataxia telangiectasia (AT) is an autosomal recessive diseaseof unknown etiology associated with cerebellar ataxia, telangiectasia,immune dysfunction, higher cancer risk, genomic instabilityand hypersensitivity to ionizing radiation. The major AT loci,AT-A and AT-C, are shown to be closely linked at chromosome11q22–q23. The most recent genetic linkage mapping andlinkage disequilibrium analysis have localized the major ATloci to a sequence of approximately 850 kb between the markersD11S1819 and D11S1818. The isolation of yeast artificial chromosomesspanning the AT region is an essential step to identify thegene or genes responsible for the mutation(s). We isolated atotal of 20 YAC clones from three independent YAC libraries,using sequence tagged sites mapped in the AT region as primersfor PCR-based YAC screening. The PCR assay for the presenceor absence of 16 different DNA markers allowed us to constructand to order four YAC contigs at the AT region. One of the contigswhich consists of the 10 YAC clones, covers about 2 Mb of DNAat the boundary between Giemsa-positive band 11q22.3 and Giemsa-negativeband 11q23.1 and includes the entire region of the major ATlocus between D11S1819 and D11S1818. Thus, the YAC contigs willfacilitate the positional cloning approach for searching transcribedsequences from the defined genomic region.     

Molecular genetic analysis of theripening-inhibitor andnon-ripening loci of tomato: A first step in genetic map-based cloning of fruit ripening genes

Ripening represents a complex developmental process unique to plants. We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype. We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato,ripening-inhibitor (rin), andnon-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process. A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning therin andnor loci, respectively. To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci. Based on this analysis, the relationship in the region spanning therin locus is estimated to be 200–300 kb/cM, while thenor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked torin andnor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA. We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains thenot locus.     

Genotype versus phenotype: conflicting results in mapping a lung tumor susceptibility locus to the G7c recombination interval in the mouse MHC class III region

Susceptibility to chemically induced lung tumorigenesis has previously been mapped to a genomic interval of 27 kb in the MHC class III region of the mouse using two H2 (a/b) intra- H2 recombinants, B10.A(1R) and B10.A(2R). Three genes are located within this interval, G7e (encoding a viral envelope protein), G7a/ Vars2 (encoding valyl-tRNA synthetase), and G7c (a gene with unknown function). A 70 kb contig, spanning the 27 kb region and extending 20 kb either side, was constructed from lambda phage libraries with genomic inserts derived from mouse strains B10.A(1R) and B10.A(2R). The region was analyzed for single-nucleotide polymorphisms, which would facilitate further fine mapping of the interval. Analysis of the expression levels of the candidate genes did not reveal any difference between B10.A(1R) and B10.A(2R). In addition, no differences were found at the sequence level in the 27 kb interval except for an A to T transition in intron 7 of G7c. A database comparison of the sequence surrounding this polymorphism did not identify any DNA-binding or enhancer consensus sequence. In conclusion, the previously observed phenotype could not be associated with or assigned to any of the candidate genes G7e, G7a/ Vars2, or G7c, nor could any of the other susceptibility loci, which have been reported to map to this region ( Cps1, Acp, Orch1, and Igis1).     

Microtubule Actin Crosslinking Factor 1 Regulates the Balbiani Body and Animal-Vegetal Polarity of the Zebrafish Oocyte

Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg.