Novel Homozygous Variants of SLC13A5 Expand the Functional Heterogeneity of a Homogeneous Syndrome of Early Infantile Epileptic Encephalopathy.

BACKGROUND: Early infantile epileptic encephalopathy 25 (EIEE25) is a distinct type of neonatal epileptic encephalopathy caused by autosomal recessive mutations in the SLC13A5 gene. SLC13A5 encodes a transmembrane sodium/citrate cotransporter required for regulating citrate entry into cells. METHODS: Four families with recessively inherited epileptic encephalopathy were sequenced by clinically accredited laboratories using commercially available epilepsy gene panels. Patients were examined by a neurologist and were clinically diagnosed with infantile epileptic encephalopathy. RESULTS: We present four families with global developmental delay, intellectual disability, and defective tooth development with four novel homozygous mutations in SLC13A5. The neurological examination showed spastic quadriplegia with increased deep tendon reflexes. Brain magnetic resonance imaging showed nonspecific signal abnormality of the bilateral hemispheric white matter. Despite similar clinical features, the conditions were based on different molecular mechanisms acting on SLC13A5 (abnormal splicing, large-scale deletions, and tandem-residue insertion). CONCLUSIONS: Our results extend the landscape of autosomal recessive inherited homozygous mutations in SLC13A5 that cause a distinctive syndrome of severe neonatal epileptic encephalopathy. Our observations confirm the homogeneity of epileptic encephalopathy and dental abnormalities as a distinct clinical marker for EIEE25 despite the heterogeneous functional and mutational background.

Wild-type S100A3 and S100A13 restore calcium homeostasis and mitigate mitochondrial dysregulation in pulmonary fibrosis patient-derived cells.

Patients with digenic S100A3 and S100A13 mutations exhibited an atypical and progressive interstitial pulmonary fibrosis, with impaired intracellular calcium homeostasis and mitochondrial dysfunction. Here we provide direct evidence of a causative effect of the mutation on receptor mediated calcium signaling and calcium store responses in control cells transfected with mutant S100A3 and mutant S100A13. We demonstrate that the mutations lead to increased mitochondrial mass and hyperpolarization, both of which were reversed by transfecting patient-derived cells with the wild type S100A3 and S100A13, or extracellular treatment with the recombinant proteins. In addition, we demonstrate increased secretion of inflammatory mediators in patient-derived cells and in control cells transfected with the mutant-encoding constructs. These findings indicate that treatment of patients' cells with recombinant S100A3 and S100A13 proteins is sufficient to normalize most of cellular responses, and may therefore suggest the use of these recombinant proteins in the treatment of this devastating disease.

ß-catenin attenuation leads to up-regulation of activating NKG2D ligands and tumor regression in BrafV600E-driven thyroid cancer cells.

Introduction: BRAFV600E mutations frequently occur in papillary thyroid cancer (PTC). ß-catenin, encoded by CTNNB1, is a key downstream component of the canonical Wnt signaling pathway and is often overexpressed in PTC. BRAFV600E-driven PTC tumors rely on Wnt/ß-catenin signaling to sustain growth and progression. Methods: In the present study, we investigated the tumorigenicity of thyroid cancer cells derived from BRAFV600E PTC mice following Ctnnb1 ablation (BVE-Ctnnb1null). Results: Remarkably, the tumorigenic potential of BVE-Ctnnb1null tumor cells was lost in nude mice. Global gene expression analysis of BVE-Ctnnb1null tumor cells showed up-regulation of NKG2D receptor activating ligands (H60a, H60b, H60c, Raet1a, Raet1b, Raet1c, Raet1d, Raet1e, and Ulbp1) and down-regulation of inhibitory MHC class I molecules H-2L and H-2K2 in BVE-Ctnnb1null tumor cells. In vitro cytotoxicity assay demonstrated that BVE-Ctnnb1wt tumor cells were resistant to NK cell-mediated cytotoxicity, whereas BVE-Ctnnb1null tumor cells were sensitive to NK cell-mediated killing. Furthermore, the overexpression of any one of these NKG2D ligands in the BVE-Ctnnb1wt cell line resulted in a significant reduction of tumor growth in nude mice. Conclusions: Our results indicate that active ß-catenin signaling inhibits NK cell-mediated immune responses against thyroid cancer cells. Targeting the ß-catenin signaling pathway may have significant therapeutic benefits for BRAF-mutant thyroid cancer by not only inhibiting tumor growth but also enhancing host immune surveillance.

The clinical utility of rapid exome sequencing in a consanguineous population.

BACKGROUND: The clinical utility of exome sequencing is now well documented. Rapid exome sequencing (RES) is more resource-intensive than regular exome sequencing and is typically employed in specialized clinical settings wherein urgent molecular diagnosis is thought to influence acute management. Studies on the clinical utility of RES have been largely limited to outbred populations. METHODS: Here, we describe our experience with rapid exome sequencing (RES) in a highly consanguineous population. Clinical settings included intensive care units, prenatal cases approaching the legal cutoff for termination, and urgent transplant decisions. RESULTS: A positive molecular finding (a pathogenic or likely pathogenic variant that explains the phenotype) was observed in 80 of 189 cases (42%), while 15 (8%) and 94 (50%) received ambiguous (variant of uncertain significance (VUS)) and negative results, respectively. The consanguineous nature of the study population gave us an opportunity to observe highly unusual and severe phenotypic expressions of previously reported genes. Clinical utility was observed in nearly all (79/80) cases with positive molecular findings and included management decisions, prognostication, and reproductive counseling. Reproductive counseling is a particularly important utility in this population where the overwhelming majority (86%) of identified variants are autosomal recessive, which are more actionable in this regard than the de novo variants typically reported by RES elsewhere. Indeed, our cost-effectiveness analysis shows compelling cost savings in the study population. CONCLUSIONS: This work expands the diversity of environments in which RES has a demonstrable clinical utility.

Identification of pharmacogenetic variants from large scale next generation sequencing data in the Saudi population.

It is well documented that drug responses are related to Absorption, Distribution, Metabolism, and Excretion (ADME) characteristics of individual patients. Several studies have identified genetic variability in pharmacogenes, that are either directly responsible for or are associated with ADME, giving rise to individualized treatments. Our objective was to provide a comprehensive overview of pharmacogenetic variation in the Saudi population. We mined next generation sequencing (NGS) data from 11,889 unrelated Saudi nationals, to determine the presence and frequencies of known functional SNP variants in 8 clinically relevant pharmacogenes (CYP2C9, CYP2C19, CYP3A5, CYP4F2, VKORC1, DPYD, TPMT and NUDT15), recommended by the Clinical Pharmacogenetics Implementation Consortium (CPIC), and collectively identified 82 such star alleles. Functionally significant pharmacogenetic variants were prevalent especially in CYP genes (excluding CYP3A5), with 10-44.4% of variants predicted to be inactive or to have decreased activity. In CYP3A5, inactive alleles (87.5%) were the most common. Only 1.8%, 0.7% and 0.7% of NUDT15, TPMT and DPYD variants respectively, were predicted to affect gene activity. In contrast, VKORC1 was found functionally, to be highly polymorphic with 53.7% of Saudi individuals harboring variants predicted to result in decreased activity and 31.3% having variants leading to increased metabolic activity. Furthermore, among the 8 pharmacogenes studied, we detected six rare variants with an aggregated frequency of 1.1%, that among several other ethnicities, were uniquely found in Saudi population. Similarly, within our cohort, the 8 pharmacogenes yielded forty-six novel variants predicted to be deleterious. Based upon our findings, 99.2% of individuals from the Saudi population carry at least one actionable pharmacogenetic variant.

Insights of Noncanonical Splice-site Variants on RNA Splicing in Patients With Congenital Hypothyroidism.

CONTEXT: Congenital hypothyroidism (CH) is caused by mutations in the genes for thyroid hormone synthesis. In our previous investigation of CH patients, approximately 53% of patients had mutations in either coding exons or canonical splice sites of causative genes. Noncanonical splice-site variants in the intron were detected but their pathogenic significance was not known. OBJECTIVE: This work aims to evaluate noncanonical splice-site variants on pre-messenger RNA (pre-mRNA) splicing of CH-causing genes. METHODS: Next-generation sequencing data of 55 CH cases in 47 families were analyzed to identify rare intron variants. The effects of variants on pre-mRNA splicing were investigated by minigene RNA-splicing assay. RESULTS: Four intron variants were found in 3 patients: solute carrier family 26 member 4 (SLC26A4) c.1544+9C>T and c.1707+94C>T in one patient, and solute carrier family 5 member 5 (SLC5A5) c.970-48G>C and c.1652-97A>C in 2 other patients. The c.1707+94C>T and c.970-48G>C caused exons 15 and 16 skipping, and exon 8 skipping, respectively. The remaining variants had no effect on RNA splicing. Furthermore, we analyzed 28 previously reported noncanonical splice-site variants (4 in TG and 24 in SLC26A4). Among them, 15 variants (~ 54%) resulted in aberrant splicing and 13 variants had no effect on RNA splicing. These data were compared with 3 variant-prediction programs (FATHMM-XF, FATHMM-MKL, and CADD). Among 32 variants, FATHMM-XF, FATHMM-MKL, and CADD correctly predicted 20 (63%), 17 (53%), and 26 (81%) variants, respectively. CONCLUSION: Two novel deep intron mutations have been identified in SLC26A4 and SLC5A5, bringing the total number of solved families with disease-causing mutations to approximately 45% in our cohort. Approximately 46% (13/28) of reported noncanonical splice-site mutations do not disrupt pre-mRNA splicing. CADD provides highest prediction accuracy of noncanonical splice-site variants.

Established and candidate transthyretin amyloidosis variants identified in the Saudi population by data mining.

BACKGROUND: Familial transthyretin (TTR) amyloidosis (ATTR) is an autosomal dominant disease with significant phenotypic heterogeneity. Its prevalence in Saudi Arabia has not previously been investigated. An existing exome variant database of Saudi individuals, sequenced to globally investigate rare diseases in the population, was mined for TTR variants and filtered for missense mutations resulting in single amino acid changes. A total of 13,906 Saudi exomes from unrelated individuals were analyzed blindly. RESULTS: Three TTR variants known to be associated with ATTR amyloidosis were identified. Additionally, three novel TTR mutations were identified. Structural analysis of the three novel variants suggests that at least two could be amyloidogenic. The most common variant associated with amyloidosis was p.Val142Ile (allele frequency 0.001). Further investigation of these variants and their translation to clinical practice may help to diagnose, monitor, and manage patients with ATTR amyloidosis. CONCLUSION: Multiple TTR variants potentially associated with systemic ATTR amyloidosis were identified in the Saudi population. Early diagnosis and intervention, facilitated by familial genetic testing of patients with ATTR amyloidosis, may benefit in the management of this disease. Early diagnosis could be enhanced through inclusion of ATTR variants in existing population-based screening programs.

ß-Catenin Attenuation Inhibits Tumor Growth and Promotes Differentiation in a BRAFV600E-Driven Thyroid Cancer Animal Model.

BRAFV600E mutation is the most frequent genetic alteration in papillary thyroid cancer (PTC). ß-Catenin (Ctnnb1) is a key downstream component of canonical Wnt signaling pathway and is frequently overexpressed in PTC. BRAF V600E-driven tumors have been speculated to rely on Wnt/ß-catenin signaling to sustain its growth, although many details remain to be elucidated. In this study, we investigated the role of ß-catenin in BrafV600E -driven thyroid cancer in a transgenic mouse model. In Braf V600E mice with wild-type (WT) Ctnnb1 (BVE-Ctnnb1WT or BVE), overexpression of ß-catenin was observed in thyroid tumors. In Braf V600E mice with Ctnnb1 knockout (BVE-Ctnnb1null), thyroid tumor growth was slowed with significant reduction in papillary architecture. This was associated with increased expression of genes involved in thyroid hormone synthesis, elevated 124iodine uptake, and serum T4. The survival of BVE-Ctnnb1null mice was increased by more than 50% during 14-month observation. Mechanistically, downregulation of MAPK, PI3K/Akt, and TGFß pathways and loss of epithelial-mesenchymal transition (EMT) were demonstrated in the BVE-Ctnnb1null tumors. Treatment with dual ß-catenin/KDM4A inhibitor PKF118-310 dramatically improved the sensitivity of BVE-Ctnnb1WT tumor cells to BRAFV600E inhibitor PLX4720, resulting in significant growth arrest and apoptosis in vitro, and tumor regression and differentiation in vivo These findings indicate that ß-catenin signaling plays an important role in thyroid cancer growth and resistance to BRAFV600E inhibitors. Simultaneously targeting both Wnt/ß-catenin and MAPK signaling pathways may achieve better therapeutic outcome in BRAFV600E inhibitor-resistant and/or radioiodine-refractory thyroid cancer.

Bi-allelic variants in HOPS complex subunit VPS41 cause cerebellar ataxia and abnormal membrane trafficking.

Membrane trafficking is a complex, essential process in eukaryotic cells responsible for protein transport and processing. Deficiencies in vacuolar protein sorting (VPS) proteins, key regulators of trafficking, cause abnormal intracellular segregation of macromolecules and organelles and are linked to human disease. VPS proteins function as part of complexes such as the homotypic fusion and vacuole protein sorting (HOPS) tethering complex, composed of VPS11, VPS16, VPS18, VPS33A, VPS39 and VPS41. The HOPS-specific subunit VPS41 has been reported to promote viability of dopaminergic neurons in Parkinson's disease but to date has not been linked to human disease. Here, we describe five unrelated families with nine affected individuals, all carrying homozygous variants in VPS41 that we show impact protein function. All affected individuals presented with a progressive neurodevelopmental disorder consisting of cognitive impairment, cerebellar atrophy/hypoplasia, motor dysfunction with ataxia and dystonia, and nystagmus. Zebrafish disease modelling supports the involvement of VPS41 dysfunction in the disorder, indicating lysosomal dysregulation throughout the brain and providing support for cerebellar and microglial abnormalities when vps41 was mutated. This provides the first example of human disease linked to the HOPS-specific subunit VPS41 and suggests the importance of HOPS complex activity for cerebellar function.

Mutation spectrum of EXT1 and EXT2 in the Saudi patients with hereditary multiple exostoses.

BACKGROUND: Hereditary Multiple Exostoses (HME), also known as Multiple Osteochondromas (MO) is a rare genetic disorder characterized by multiple benign cartilaginous bone tumors, which are caused by mutations in the genes for exostosin glycosyltransferase 1 (EXT1) and exostosin glycosyltransferase 2 (EXT2). The genetic defects have not been studied in the Saudi patients. AIM OF STUDY: We investigated mutation spectrum of EXT1 and EXT2 in 22 patients from 17 unrelated families. METHODS: Genomic DNA was extracted from peripheral leucocytes. The coding regions and intron-exon boundaries of both EXT1 and EXT2 genes were screened for mutations by PCR-sequencing analysis. Gross deletions were analyzed by MLPA analysis. RESULTS: EXT1 mutations were detected in 6 families (35%) and 3 were novel mutations: c.739G > T (p. E247*), c.1319delG (p.R440Lfs*4), and c.1786delA (p.S596Afs*25). EXT2 mutations were detected in 7 families (41%) and 3 were novel mutations: c.541delG (p.D181Ifs*89), c.583delG (p.G195Vfs*75), and a gross deletion of approximately 10 kb including promoter and exon 1. Five patients from different families had no family history and carried de novo mutations (29%, 5/17). No EXT1 and EXT2 mutations were found in the remaining four families. In total, EXT1 and EXT2 mutations were found in 77% (13/17) of Saudi HME patients. CONCLUSION: EXT1 and EXT2 mutations contribute significantly to the pathogenesis of HME in the Saudi population. In contrast to high mutation rate in EXT 1 (65%) and low mutation rate in EXT2 (25%) in other populations, the frequency of EXT2 mutations are much higher (41%) and comparable to that of EXT1 among Saudi patients. De novo mutations are also common and the six novel EXT1/EXT2 mutations further expands the mutation spectrum of HME.

Molecular Analysis of CYP27B1 Mutations in Vitamin D-Dependent Rickets Type 1A: c.590G > A (p.G197D) Missense Mutation Causes a RNA Splicing Error.

CONTEXT: Vitamin D-dependent rickets type 1A (VDDR1A) is a rare autosomal recessively inherited disorder due to loss-of-function mutations in the CYP27B1 gene. CYP27B1 encodes an enzyme of 25-hydroxyvitamin D-1α-hydroxylase for converting inactive 25-OHD to biologically active 1,25-(OH)2D. OBJECTIVE: To identify underlying genetic defects in patients with VDDR1A. METHODS: Twelve patients from 7 Turkish and 2 Saudi families were investigated. The coding exons and intron-exon boundaries of the CYP27B1 gene were amplified by Polymerase Chain Reaction (PCR) from peripheral lymphocyte DNA. PCR products were directly sequenced. The consequences of c.590G > A mutation were analyzed by in silico and functional analysis. RESULTS: CYP27B1 mutations were identified in all the patients. Two novel mutations were identified in two separate families: c.171delG (family 7) and c.398_400dupAAT (family 8). The intra-exon deletion of c.171delG resulted in a frameshift and premature stop codon 20 amino acids downstream from the mutation (p.L58Cfs∗20). The intra-exon duplication of c.398_400dupAAT generated a premature stop codon at the mutation site (p.W134∗). A missense c.590G > A (p.G197D) mutation was found in a patient from family 4 and caused a defect in pre-mRNA splicing. As a result, two populations of transcripts were detected: the majority of them with intron 3 retention (83%), and the minority (17%) being properly spliced transcripts with about 16% of wild-type enzymatic activity. The remaining nine patients from six families carried a previously reported c.1319_1325dupCCCACCC (F443Pfs∗24) mutation. Clinically, all the patients need continued calcitriol treatment, which was consistent with inactivation of 25-hydroxy vitamin D1α-hydroxylase activity. CONCLUSION: Two novel frameshift CYP27B1 mutations were identified and predicted to inactivate 25-hydroxyvitamin D-1α-hydroxylase. The loss of enzymatic activity by c.590G > A missense mutation was mainly caused by aberrant pre-mRNA splicing.

NOVEL VDR MUTATIONS IN PATIENTS WITH VITAMIN D-DEPENDENT RICKETS TYPE 2A: A MILD DISEASE PHENOTYPE CAUSED BY A NOVEL CANONICAL SPLICE-SITE MUTATION.

Objective: Vitamin D-dependent rickets type 2A (VDDR2A) is a rare autosomal recessive disorder caused by mutations in the vitamin D receptor gene (VDR), leading to end-organ resistance to 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). The objective of this study was to investigate VDR mutations in 11 patients from 8 Turkish-Arab families. Methods: All coding exons and intron-exon boundaries of the VDR gene were amplified by polymerase chain reaction from peripheral leukocyte DNA and sequenced. The effect of splice-site mutations on mRNA splicing was evaluated by a customized VDR mini-gene assay. Results: Homozygous VDR mutations were found in all the patients, including four novel mutations: c.473G>T (p.R158L), c.1-4A>G (IVS3-2A>G), c.755+1G>T, and c.352_356delGACAG (p.D118Sfs*7). The c.1-4A>G mutation was located in the canonical splice acceptor site and 4 base pairs from the original ATG start codon. The mutation resulted in both complete (60% of transcripts) and partial exon 4 skipping (15% of transcripts). The latter was due to activation of a cryptic splice acceptor site and did not disrupt the open reading frame. Both c.755+1G>T and c.352_356delGACAG resulted in frameshifts and a premature stop codon. Clinically, all the patients required continued treatment, except for patient IV-3, who presented with alopecia, hypocalcemia, and increased 1,25(OH)2D3 at 1.5 years of age as a result of the c.1-4A>G mutation. He stopped taking medication at 6 years of age and still maintained normal height and biochemical profile. Conclusion: We have identified four novel VDR mutations. Although canonical splice-site mutations cause premRNA splicing errors that usually lead to a severe disease phenotype, mild disease can also occur due to activation of a cryptic splice site. Abbreviations: 1,25(OH)2D3 = 1,25-dihydroxyvitamin D3 (calcitriol); 25OHD3 = 25-hydroxyvitamin D3; PCR = polymerase chain reaction; PTH = parathyroid hormone; VDDR2A = vitamin D-dependent rickets type 2A; VDR = vitamin D receptor.

Mutation of SGK3, a Novel Regulator of Renal Phosphate Transport, Causes Autosomal Dominant Hypophosphatemic Rickets.

CONTEXT: Hypophosphatemic rickets (HR) is a group of rare hereditary renal phosphate wasting disorders caused by mutations in PHEX, FGF23, DMP1, ENPP1, CLCN5, SLC9A3R1, SLC34A1, or SLC34A3. OBJECTIVE: A large kindred with 5 HR patients was recruited with dominant inheritance. The study was undertaken to investigate underlying genetic defects in HR patients. DESIGN: Patients and their family members were initially analyzed for PHEX and FGF23 mutations using polymerase chain reaction sequencing and copy number analysis. Exome sequencing was subsequently performed to identify novel candidate genes. RESULTS: PHEX and FGF23 mutations were not detected in the patients. No copy number variation was observed in the genome using CytoScan HD array analysis. Mutations in DMP1, ENPP1, CLCN5, SLC9A3R1, SLC34A1, or SLC34A3 were also not found by exome sequencing. A novel c.979-96 T>A mutation in the SGK3 gene was found to be strictly segregated in a heterozygous pattern in patients and was not present in normal family members. The mutation is located 1 bp downstream of a highly conserved adenosine branch point, resulted in exon 13 skipping and in-frame deletion of 29 amino acids, which is part of the protein kinase domain and contains a Thr-320 phosphorylation site that is required for its activation. Protein tertiary structure modelling showed significant structural change in the protein kinase domain following the deletion. CONCLUSIONS: The c.979-96 T>A splice mutation in the SGK3 gene causes exon 13 skipping and deletion of 29 amino acids in the protein kinase domain. The SGK3 mutation may cause autosomal dominant HR.

Functional analysis of 22 splice-site mutations in the PHEX, the causative gene in X-linked dominant hypophosphatemic rickets.

CONTEXT: X-linked hypophosphatemic rickets (XLH) is caused by inactivating mutations in the PHEX gene and is the most common form of hereditary rickets. The splice-site mutations account for 17% of all reported PHEX mutations. The functional consequence of these splice-site mutations has not been systemically investigated. OBJECTIVE: The current study was undertaken to functionally annotate previously reported 22 splice-site mutations in the PHEX gene. METHODS: PHEX mini-genes with different splice-site mutations were created by site-directed mutagenesis and expressed in HEK293 cells. The mRNA transcripts were analyzed by RT-PCR, cloning, and sequencing. RESULTS: These splicing mutations led to a variety of consequences, including exon skipping, intron retention, and activation of cryptic splice sites. Among 22 splice-site mutations, exon skipping was the most common event accounting for 73% (16/22). Non-canonical splice-site mutations could result in splicing errors to the same extent as canonical splice-site mutations such as c.436+3G>C, c.436+4A>C, c.436+6T>C, c.437-3C>G, c.850-3C>G, c.1080-3C>A, c.1482+5G>C, c.1586+6T>C, c.1645+5G>A, c.1645+6T>C, c.1701-16T>A, c.1768+5G>A, and c.1899+5G>A. Interestingly, non-canonical (c.436+6T>C and c.1586+6T>C) and canonical splice-site mutations (c.1769-1G>C) could generate partial splicing errors (both wild-type and mutant transcripts were detected), resulting in incomplete inactivation of PHEX gene, which may explain the mild disease phenotype reported previously, providing evidence of genotype-phenotype correlation. c.1645C>T (p.R549*) had no impact on pre-mRNA splicing although it is located next to canonical splice donor site GT. CONCLUSIONS: Exon skipping is the most common outcome due to splice-site mutations. Both canonical and non-canonical splice-site mutations can result in either severe or mild RNA splicing defects, contributing to phenotype heterogeneity. Non-canonical splice-site mutations should not be overlooked in genetic screening especially those located within 50 bp from canonical splice site.

An atypical pulmonary fibrosis is associated with co-inheritance of mutations in the calcium binding protein genes S100A3 and S100A13.

BACKGROUND: Pulmonary fibrosis is one of the leading indications for lung transplantation. The disease, which is of unknown aetiology, can be progressive, resulting in distortion of the extracellular matrix (ECM), inflammation, fibrosis and eventual death. METHODS: 13 patients born to consanguineous parents from two unrelated families presenting with interstitial lung disease were clinically investigated. Nine patients developed respiratory failure and subsequently died. Molecular genetic investigations were performed on patients' whole blood or archived tissues, and cell biological investigations were performed on patient-derived fibroblasts. RESULTS: The combination of a unique pattern of early-onset lung fibrosis (at 12-15 years old) with distinctive radiological findings, including 1) traction bronchiectasis, 2) intralobular septal thickening, 3) shrinkage of the secondary pulmonary lobules mainly around the bronchovascular bundles and 4) early type 2 respiratory failure (elevated blood carbon dioxide levels), represents a novel clinical subtype of familial pulmonary fibrosis. Molecular genetic investigation of families revealed a hypomorphic variant in S100A3 and a novel truncating mutation in S100A13, both segregating with the disease in an autosomal recessive manner. Family members that were either heterozygous carriers or wild-type normal for both variants were unaffected. Analysis of patient-derived fibroblasts demonstrated significantly reduced S100A3 and S100A13 expression. Further analysis demonstrated aberrant intracellular calcium homeostasis, mitochondrial dysregulation and differential expression of ECM components. CONCLUSION: Our data demonstrate that digenic inheritance of mutations in S100A3 and S100A13 underlie the pathophysiology of pulmonary fibrosis associated with a significant reduction of both proteins, which suggests a calcium-dependent therapeutic approach for management of the disease.

Autosomal recessive ADCY5-Related dystonia and myoclonus: Expanding the genetic spectrum of ADCY5-Related movement disorders.

INTRODUCTION: ADCY5-related hyperkinesia encompasses a heterogeneous group of phenotypes, including paroxysmal chorea, myoclonus, and dystonia. The disease is attributed to mutations of ADCY5, which encodes an adenylate cyclase enzyme. The disease can occur in a sporadic or familial pattern. With exception of one study, all reports on familial ADCY5-related hyperkinesia were associated with an autosomal dominant inheritance. Herein, we describe a native Arabian Bedouin family with an autosomal recessive ADCY5-related disorder and expand the genotypic and phenotypic spectrum of this disorder. METHODS: The pedigree included 4 generations of a family with 6 affected individuals. The patients were examined clinically and radiologically. Homozygosity mapping and Whole Exome Sequencing (WES) were used to identify a variant, predicted to be pathogenic, which segregated with disease in this family. RESULTS: All patients presented with early-onset dystonia and myoclonus. The patients had delayed motor and language milestones, axial hypotonia, severe anxiety, social phobia, and isolation. One patient had dilated cardiomyopathy. WES of one affected individual revealed a novel homozygous missense mutation (c.1762G > A, p.D588N) of ADCY5, that segregated with disease in an autosomal recessive manner, and was absent in more than 1000 ethnically-matched chromosomes. The mutation replaces a highly conserved nucleotide and is predicted to be deleterious. CONCLUSION: This study reports the second family with autosomal recessive childhood-onset ADCY5-related disorder and expands our understanding of phenotype/genotype correlations of this disorder.

Pattern Recognition Receptor Polymorphisms as Predictors of Oxaliplatin Benefit in Colorectal Cancer.

BACKGROUND: Constitutional loss of function (LOF) single nucleotide polymorphisms (SNPs) in pattern recognition receptors FPR1, TLR3, and TLR4 have previously been reported to predict oxaliplatin benefit in colorectal cancer. Confirmation of this association could substantially improve patient stratification. METHODS: We performed a retrospective biomarker analysis of the Short Course in Oncology Therapy (SCOT) and COIN/COIN-B trials. Participant status for LOF variants in FPR1 (rs867228), TLR3 (rs3775291), and TLR4 (rs4986790/rs4986791) was determined by genotyping array or genotype imputation. Associations between LOF variants and disease-free survival (DFS) and overall survival (OS) were analyzed by Cox regression, adjusted for confounders, using additive, dominant, and recessive genetic models. All statistical tests were two-sided. RESULTS: Our validation study populations included 2929 and 1948 patients in the SCOT and COIN/COIN-B cohorts, respectively, of whom 2728 and 1672 patients had functional status of all three SNPs determined. We found no evidence of an association between any SNP and DFS in the SCOT cohort, or with OS in either cohort, irrespective of the type of model used. This included models for which an association was previously reported for rs867228 (recessive model, multivariable-adjusted hazard ratio [HR] for DFS in SCOT = 1.19, 95% confidence interval [CI] = 0.99 to 1.45, P = .07; HR for OS in COIN/COIN-B = 0.92, 95% CI = 0.63 to 1.34, P = .66), and rs4986790 (dominant model, multivariable-adjusted HR for DFS in SCOT = 0.86, 95% CI = 0.65 to 1.13, P = .27; HR for OS in COIN/COIN-B = 1.08, 95% CI = 0.90 to 1.31, P = .40). CONCLUSION: In this prespecified analysis of two large clinical trials, we found no evidence that constitutional LOF SNPs in FPR1, TLR3, or TLR4 are associated with differential benefit from oxaliplatin. Our results suggest these SNPs are unlikely to be clinically useful biomarkers.

De novo truncating variants in WHSC1 recapitulate the Wolf-Hirschhorn (4p16.3 microdeletion) syndrome phenotype.

PURPOSE: Wolf-Hirschhorn syndrome (WHS) is a genomic disorder with a recognizable dysmorphology profile caused by hemizygosity at 4p16.3. Previous attempts have failed to map the minimal critical locus to a single gene, leaving open the possibility that the core phenotypic components of the syndrome are caused by the combined haploinsufficiency of multiple genes. METHODS: Clinical exome sequencing and "reverse" phenotyping. RESULTS: We identified two patients with de novo truncating variants in WHSC1, which maps to the WHS critical locus. The phenotype of these two individuals is consistent with WHS, which suggests that haploinsufficiency of WHSC1 is sufficient to recapitulate the core phenotype (characteristic facies, and growth and developmental delay) of this classic microdeletion syndrome. CONCLUSION: Our study expands the list of microdeletion syndromes that are solved at the single-gene level, and establishes WHSC1 as a disease gene in humans. Given the severe nature of the reported variants, the full phenotypic expression of WHSC1 may be further expanded by future reports of milder variants.