Tiancheng Wang

Concept and design of a genome-wide association genotyping array tailored for transplantation-specific studies

BackgroundIn addition to HLA genetic incompatibility, non-HLA difference between donor and recipients of transplantation leading to allograft rejection are now becoming evident. We aimed to create a unique genome-wide platform to facilitate genomic research studies in transplant-related studies. We designed a genome-wide genotyping tool based on the most recent human genomic reference datasets, and included customization for known and potentially relevant metabolic and pharmacological loci relevant to transplantation.MethodsWe describe here the design and implementation of a customized genome-wide genotyping array, the ‘TxArray’, comprising approximately 782,000 markers with tailored content for deeper capture of variants across HLA, KIR, pharmacogenomic, and metabolic loci important in transplantation. To test concordance and genotyping quality, we genotyped 85 HapMap samples on the array, including eight trios.ResultsWe show low Mendelian error rates and high concordance rates for HapMap samples (average parent-parent-child heritability of 0.997, and concordance of 0.996). We performed genotype imputation across autosomal regions, masking directly genotyped SNPs to assess imputation accuracy and report an accuracy of >0.962 for directly genotyped SNPs. We demonstrate much higher capture of the natural killer cell immunoglobulin-like receptor (KIR) region versus comparable platforms. Overall, we show that the genotyping quality and coverage of the TxArray is very high when compared to reference samples and to other genome-wide genotyping platforms.ConclusionsWe have designed a comprehensive genome-wide genotyping tool which enables accurate association testing and imputation of ungenotyped SNPs, facilitating powerful and cost-effective large-scale genotyping of transplant-related studies.

Copy Number Variations in CTNNA3 and RBFOX1 Associate with Pediatric Food Allergy

Food allergy is a significant public health concern, especially among children. Previous candidate gene studies suggested a few susceptibility loci for food allergy, but no study investigated the contribution of copy number variations (CNVs) to food allergy on a genome-wide scale. To investigate the genetics of food allergy, we performed CNV assessment using high-resolution genome-wide single nucleotide polymorphism arrays. CNV calls from a total of 357 cases with confirmed food allergy and 3980 controls were analyzed within a discovery cohort, followed by a replication analysis composed of 167 cases and 1573 controls. We identified that CNVs in CTNNA3 were significantly associated with food allergy in both the discovery cohort and the replication cohort. Of particular interest, CTNNA3 CNVs hit exons or intron regions rich in histone marker H3K4Me1. CNVs in a second gene (RBFOX1) showed a significant association (p = 7.35 × 10−5) with food allergy at the genome-wide level in our meta-analysis of the European ancestry cohorts. The presence of these CNVs was confirmed by quantitative PCR. Furthermore, knockdown of CTNNA3 resulted in upregulation of CD63 and CD203c in mononuclear cells upon PMA stimulation, suggesting a role in sensitization to allergen. We uncovered at least two plausible genes harboring CNV loci that are enriched in pediatric patients with food allergies. The novel gene candidates discovered in this study by genome-wide CNV analysis are compelling drug and diagnostic targets for food allergy.

Expanding the SPECC1L mutation phenotypic spectrum to include Teebi hypertelorism syndrome

Teebi hypertelorism syndrome is a rare autosomal dominant disorder that has eluded a molecular etiology since first described in 1987. Here we report on two unrelated families with a Teebi hypertelorism‐like syndrome and Teebi hypertelorism phenotype who have missense mutations in Sperm Antigen With Calponin Homology And Coiled‐Coil Domains (SPECC1L), previously associated with oblique facial clefting and Opitz G/BBB syndrome. The first patient and his affected mother were previously‐reported by Hoffman et al. in this journal as a new syndrome resembling Teebi hypertelorism and Aarskog syndromes in 2007. This patient had hypertelorism, sagittal and coronal craniosynostosis, ptosis, natal teeth, unusual umbilicus, shawl scrotum, small hands, and feet, with grossly normal development. Our second patient had classic Teebi hypertelorism syndrome with hypertelorism and a giant umbilical hernia. Patient one and his affected mother had a c.1260G>C:p.E420D variant and patient two had a de novo c.1198_1203delATACAC:p.I400_H401del variant in SPECC1L. We review the phenotypic findings in the previously‐published Teebi hypertelorism syndrome patients, and the Opitz G/BBB patients with SPECC1L mutations. In addition we emphasize the findings of aortic root dilation and craniosynostosis in these patients, which should be considered in their management.

CNV Analysis Associates AKNAD1 with Type-2 Diabetes in Jordan Subpopulations.

Previous studies have identified a number of single nucleotide polymorphisms (SNPs) associated with type-2 diabetes (T2D), but copy number variation (CNV) association has rarely been addressed, especially in populations from Jordan. To investigate CNV associations for T2D in populations in Jordan, we conducted a CNV analysis based on intensity data from genome-wide SNP array, including 34 T2D cases and 110 healthy controls of Chechen ethnicity, as well as 34 T2D cases and 106 healthy controls of Circassian ethnicity. We found a CNV region in protein tyrosine phosphatase receptor type D (PTPRD) with significant association with T2D. PTPRD has been reported to be associated with T2D in genome-wide association studies (GWAS). We additionally identified 16 CNV regions associated with T2D which overlapped with gene exons. Of particular interest, a CNV region in the gene AKNA Domain Containing 1 (AKNAD1) surpassed the experiment-wide significance threshold. Endoplasmic reticulum (ER)-related pathways were significantly enriched among genes which are predicted to be functionally associated with human or mouse homologues of AKNAD1. This is the first CNV analysis of a complex disease in populations of Jordan. We identified and experimentally validated a significant CNVR in gene AKNAD1 associated with T2D.

Pathogenic variant in EPHB4 results in central conducting lymphatic anomaly.

Central conducting lymphatic anomaly (CCLA) is one of the complex lymphatic anomalies characterized by dilated lymphatic channels, lymphatic channel dysmotility and distal obstruction affecting lymphatic drainage. We performed whole exome sequencing (WES) of DNA from a four-generation pedigree and examined the consequences of the variant by transfection of mammalian cells and morpholino and rescue studies in zebrafish. WES revealed a heterozygous mutation in EPHB4 (RefSeq NM_004444.4; c.2334 + 1G>C) and RNA-Seq demonstrated that the EPHB4 mutation destroys the normal donor site, which leads to the use of a cryptic splice donor that results in retention of the intervening 12-bp intron sequence. Transient co-expression of the wild-type and mutant EPHB4 proteins showed reduced phosphorylation of tyrosine, consistent with a loss-of-function effect. Zebrafish ephb4a morpholino resulted in vessel misbranching and deformities in the lymphatic vessel development, indicative of possible differentiation defects in lymphatic vessels, mimicking the lymphatic presentations of the patients. Immunoblot analysis using zebrafish lysates demonstrated over-activation of mTORC1 as a consequence of reduced EPHB4 signaling. Strikingly, drugs that inhibit mTOR signaling or RAS-MAPK signaling effectively rescued the misbranching phenotype in a comparable manner. Moreover, knock-in of EPHB4 mutation in HEK293T cells also induced mTORC1 activity. Our data demonstrate the pathogenicity of the identified EPHB4 mutation as a novel cause of CCLA and suggesting that ERK inhibitors may have therapeutic benefits in such patients with complex lymphatic anomalies.

Fasoracetam in adolescents with ADHD and glutamatergic gene network variants disrupting mGluR neurotransmitter signaling

The glutamatergic neurotransmitter system may play an important role in attention-deficit hyperactivity disorder (ADHD). This 5-week, open-label, single-blind, placebo-controlled study reports the safety, pharmacokinetics and responsiveness of the metabotropic glutamate receptor (mGluR) activator fasoracetam (NFC-1), in 30 adolescents, age 12–17 years with ADHD, harboring mutations in mGluR network genes. Mutation status was double-blinded. A single-dose pharmacokinetic profiling from 50–800 mg was followed by a single-blind placebo at week 1 and subsequent symptom-driven dose advancement up to 400 mg BID for 4 weeks. NFC-1 treatment resulted in significant improvement. Mean Clinical Global Impressions-Improvement (CGI-I) and Severity (CGI-S) scores were, respectively, 3.79 at baseline vs. 2.33 at week 5 (P < 0.001) and 4.83 at baseline vs. 3.86 at week 5 (P < 0.001). Parental Vanderbilt scores showed significant improvement for subjects with mGluR Tier 1 variants (P < 0.035). There were no differences in the incidence of adverse events between placebo week and weeks on active drug. The trial is registered at https://clinicaltrials.gov/ct2/show/study/NCT02286817.Stimulant drugs are most commonly used to treat ADHD. Here, the authors demonstrate that in adolescents with ADHD who also have genetic variation in genes impacting metabotropic glutamate signaling, the non-stimulant mGluR activator fasoracetam is well tolerated and may be beneficial in alleviating symptoms of this disease.

Genome-wide association study reveals two loci for serum magnesium concentrations in European-American children.

Magnesium ions are essential to the basic metabolic processes in the human body. Previous genetic studies indicate that serum magnesium levels are highly heritable, and a few genetic loci have been reported involving regulation of serum magnesium in adults. In this study, we examined if additional loci influence serum magnesium levels in children. We performed a genome-wide association study (GWAS) on 2,267 European-American children genotyped on the Illumina HumanHap550 or Quad610 arrays, sharing over 500,000 markers, as the discovery cohort and 257 European-American children genotyped on the Illumina Human OmniExpress arrays as the replication cohort. After genotype imputation, the strongest associations uncovered were with imputed SNPs residing within the FGFR2 (rs1219515, P = 1.1 × 10−5) and PAPSS2 (rs1969821, P = 7.2 × 10−6) loci in the discovery cohort, both of which were robustly replicated in our independent patient cohort (rs1219515, P = 3.5 × 10−3; rs1969821, P = 1.2 × 10−2). The associations at the FGFR2 locus were also weakly replicated in a dataset from a previous GWAS of serum magnesium in European adults. Our results indicate that FGFR2 and PAPSS2 may play an important role in the regulation of magnesium homeostasis in children of European-American ancestry.

Heterozygous Mutations in TBX1 as a Cause of Isolated Hypoparathyroidism

Context Most cases of autosomal dominant isolated hypoparathyroidism are caused by gain-of-function mutations in CASR or GNA11 or dominant negative mutations in GCM2 or PTH. Objective To identify the genetic etiology for dominantly transmitted isolated hypoparathyroidism in two multigenerational families with 14 affected family members. Methods We performed whole exome sequencing of DNA from two families and examined the consequences of mutations by minigene splicing assay. Results We discovered disease-causing mutations in both families. A splice-altering mutation in TBX1 (c.1009+1G>C) leading to skipping of exon 8 (101 bp) was identified in 10 affected family members and five unaffected subjects of family A, indicating reduced penetrance for this point mutation. In a second family from France (family B), we identified another splice-altering mutation (c.1009+2T>C) adjacent to the mutation identified in family A that results in skipping of the same exon; two subjects in family B had isolated hypoparathyroidism, whereas a third subject manifested the clinical triad of the 22q11.2 deletion syndrome, indicative of variable expressivity. Conclusions We report evidence that heterozygous TBX1 mutations can cause isolated hypoparathyroidism. This study adds knowledge to the increasingly expanding list of causative and candidate genes in isolated hypoparathyroidism.

Heterozygous Deletion Impacting SMARCAD1 in the Original Kindred with Absent Dermatoglyphs and Associated Features (Baird, 1964)

&NA; In 1964, Baird described a family with adermatoglyphia, facial milia, and skin fragility. Using whole exome sequencing, genotyping, and Sanger sequencing, we identified a 116‐kb heterozygous deletion involving exons 1‐9 of SMARCAD1 in descendants of this kindred. This contrasts with point mutations within exon 9 in all other reported families.

Early Infantile Epileptic Encephalopathy in an STXBP1 Patient with Lactic Acidemia and Normal Mitochondrial Respiratory Chain Function

A wide range of clinical findings have been associated with mutations in Syntaxin Binding Protein 1 (STXBP1), including multiple forms of epilepsy, nonsyndromic intellectual disability, and movement disorders. STXBP1 mutations have recently been associated with mitochondrial pathology, although it remains unclear if this phenotype is a part of the core feature for this gene disorder. We report a 7-year-old boy who presented for diagnostic evaluation of intractable epilepsy, episodic ataxia, resting tremor, and speech regression following a period of apparently normal early development. Mild lactic acidemia was detected on one occasion at the time of an intercurrent illness. Due to the concern for mitochondrial disease, ophthalmologic evaluation was performed that revealed bilateral midperiphery pigmentary mottling. Optical coherence tomography (OCT) testing demonstrated a bilaterally thickened ganglion cell layer in the perifovea. Skeletal muscle biopsy analysis showed no mitochondrial abnormalities or respiratory chain dysfunction. Exome sequencing identified a de novo c.1651C>T (p.R551C) mutation in STXBP1. Although mitochondrial dysfunction has been reported in some individuals, our proband had only mild lactic acidemia and no skeletal muscle tissue evidence of mitochondrial disease pathology. Thus, mitochondrial dysfunction is not an obligate feature of STXBP1 disease.