Intellectual disability associated with a homozygous missense mutation in THOC6

BACKGROUND: We recently described a novel autosomal recessive neurodevelopmental disorder with intellectual disability in four patients from two related Hutterite families. Identity-by-descent mapping localized the gene to a 5.1 Mb region at chromosome 16p13.3 containing more than 170 known or predicted genes. The objective of this study was to identify the causative gene for this rare disorder. METHODS AND RESULTS: Candidate gene sequencing followed by exome sequencing identified a homozygous missense mutation p.Gly46Arg, in THOC6. No other potentially causative coding variants were present within the critical region on chromosome 16. THOC6 is a member of the THO/TREX complex which is involved in coordinating mRNA processing with mRNA export from the nucleus. In situ hybridization showed that thoc6 is highly expressed in the midbrain and eyes. Cellular localization studies demonstrated that wild-type THOC6 is present within the nucleus as is the case for other THO complex proteins. However, mutant THOC6 was predominantly localized to the cytoplasm, suggesting that the mutant protein is unable to carry out its normal function. siRNA knockdown of THOC6 revealed increased apoptosis in cultured cells. CONCLUSION: Our findings associate a missense mutation in THOC6 with intellectual disability, suggesting the THO/TREX complex plays an important role in neurodevelopment

Mutations in LAMA1 Cause Cerebellar Dysplasia and Cysts with and without Retinal Dystrophy

Cerebellar dysplasia with cysts (CDC) is an imaging finding typically seen in combination with cobblestone cortex and congenital muscular dystrophy in individuals with dystroglycanopathies. More recently, CDC was reported in seven children without neuromuscular involvement (Poretti-Boltshauser syndrome). Using a combination of homozygosity mapping and whole-exome sequencing, we identified biallelic mutations in LAMA1 as the cause of CDC in seven affected individuals (from five families) independent from those included in the phenotypic description of Poretti-Boltshauser syndrome. Most of these individuals also have high myopia, and some have retinal dystrophy and patchy increased T2-weighted fluid-attenuated inversion recovery (T2/FLAIR) signal in cortical white matter. In one additional family, we identified two siblings who have truncating LAMA1 mutations in combination with retinal dystrophy and mild cerebellar dysplasia without cysts, indicating that cysts are not an obligate feature associated with loss of LAMA1 function. This work expands the phenotypic spectrum associated with the lamininopathy disorders and highlights the tissue-specific roles played by different laminin-encoding genes

Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1. yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS

An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge

There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. RESULTS: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. CONCLUSIONS: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups

Genetic mechanisms of neurodegeneration

Neurodegenerative diseases are characterized by accelerated and/or inappropriate neuronal loss which is thought to be caused by genetic and environmental factors. At present predisposing factors remain unknown and effective therapies are not available for many of these diseases. However, recent work has described at least 4 pathogenic mechanisms. (1) Oxidative stress: mutations in the superoxide dismutase gene cause approximately 1% of ALS cases. (2) Neurofilament abnormalities: neurofilament overexpression causes an ALS-like phenotype in mice, while pathologically, neurofilament accumulation is observed in patients. (3) Deregulation of apoptosis: deletions in apoptosis-regulatory genes are present in spinal muscular atrophy. (4) Expansion of trinucleotide repeats---expansions are responsible for many neurodegenerative diseases including fragile X and Huntington's disease.My work involved the investigation of these mechanisms in the pathogenesis of ALS and Parkinson's disease. Identification of molecular defects in genes regulating these pathways would provide direct evidence linking these mechanisms to neurodegeneration. Such evidence was not found. Mutations in the Mn SOD and catalase genes were absent in both disease populations. The Cu/Zn SOD gene was not mutated in Parkinson's patients. A novel allele at the NEFH KSP repeat locus was identified in Guam and Kii ALS patients but its presence in non-affected Guam and Japanese individuals eliminated it as a causative mutation. The same allele, absent in non-Asian Pacific populations, was identified in 7 affected individuals of an atypical Puerto Rican Parkinson-dystonia family. Further studies are necessary to determine whether the disease in this family is associated with this locus.Results from the study of the SMA- and SBMA-causing mutations in ALS, revealed the presence of diagnostic errors within the ALS population. While not unexpected, the significant differences in prognosis between SMA or SBMA and ALS, make this differentiation important. Caucasian SBMA was found to be due to new mutations and not predisposing or founder chromosomes as reported in Japanese SBMA and other trinucleotide repeat diseases.While the etiology of neurodegenerative disease is complex, my work has led to a significant increase in our understanding of this complex group of disorders. Much additional work needs to be done so that accurate diagnoses can be made and targeted effective treatments developed

Biochemical and molecular analysis of monoamine oxidase in alcoholics, high risk subjects and low risk controls

Alcoholism is a prevalent multifactorial disease with both genetic and environmental components. Monoamine oxidase (MAO) has been proposed as a susceptibility marker for familial alcoholism but consistent evidence of either specific MAO variants in alcoholics or allelic segregation in at-risk families has not been presented. Two structural genes on the X chromosome encode two forms of the enzyme, MAO-A and MAO-B. Kinetic constants for platelet MAO-B and restriction fragment length polymorphisms for MAO-A were determined in alcoholics with multigenerational family histories of alcoholism, high risk relatives of familial alcoholics and low risk controls with no family history of alcoholism. Mean elevated levels of MAO-B deamination were observed in alcoholics and high risk individuals. Alcoholic and high risk individuals did not differ from non-alcoholics with respect to MAO-B tryptamine affinity or MAO-A polymorphisms. Significant non-genetic factors influence MAO-B activity. MAO variants are unlikely to define a genetic predisposition to alcoholism

The role of serotonin receptor alleles and environmental stressors in the development of post-concussive symptoms after pediatric mild traumatic brain injury

Aim: To determine whether post-injury depressive symptoms, and pre-injury major life stressors and genetic factors (HTR1A C(-1019)G alleles; rs6295) are more common in children with mild traumatic brain injury (mTBI) who develop postconcussion syndrome (PCS) symptoms compared with children with asymptomatic mTBI. Method: This was a cross-sectional study of 47 symptomatic children (32 males, 15 females; mean age 14y [SD 3y 3mo]) who experienced post-concussive symptoms for 7 or more days and 42 asymptomatic children (26 males 16 females; mean age 13y 6mo [SD 3y 1mo]) after mTBI. Outcome measures were the Postconcussion Symptoms Inventory (PCSI), the Children's Depression Inventory (CDI), standard questionnaire of previous life events, and buccal DNA analysis to determine genotype and allele frequencies for the HTR1A C(-1019)G polymorphism. Results: Depressive symptoms were uncommon. CDI scores did not differ between groups. Allelic and genotypic frequencies for HTR1A C(-1019)G were similar in both groups. Symptomatic children continued to have elevated PCS scores compared with asymptomatic children 1.72 (SD 0.69) years later and had experienced significantly more life stressors (Wald (1)=8.51, p=0.004). Interpretation: HTR1A polymorphisms do not differ in children with PCS. Children who have experienced more significant life stresses are more likely to develop PCS symptoms after mTBI

Homozygous Deletion of the Very Low Density Lipoprotein Receptor Gene Causes Autosomal Recessive Cerebellar Hypoplasia with Cerebral Gyral Simplification

An autosomal recessive syndrome of nonprogressive cerebellar ataxia and mental retardation is associated with inferior cerebellar hypoplasia and mild cerebral gyral simplification in the Hutterite population. An identity-by-descent mapping approach using eight patients from three interrelated Hutterite families localized the gene for this syndrome to chromosome region 9p24. Haplotype analysis identified familial and ancestral recombination events and refined the minimal region to a 2-Mb interval between markers D9S129 and D9S1871. A 199-kb homozygous deletion encompassing the entire very low density lipoprotein receptor (VLDLR) gene was present in all affected individuals. VLDLR is part of the reelin signaling pathway, which guides neuroblast migration in the cerebral cortex and cerebellum. To our knowledge, this syndrome represents the first human lipoprotein receptor malformation syndrome and the second human disease associated with a reelin pathway defect

Hnrnpul1 controls transcription, splicing, and modulates skeletal and limb development in vivo

Mutations in RNA-binding proteins can lead to pleiotropic phenotypes including craniofacial, skeletal, limb, and neurological symptoms. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in nucleic acid binding, transcription, and splicing through direct binding to DNA and RNA, or through interaction with other proteins in the spliceosome. We show a developmental role for Hnrnpul1 in zebrafish, resulting in reduced body and fin growth and missing bones. Defects in craniofacial tendon growth and adult-onset caudal scoliosis are also seen. We demonstrate a role for Hnrnpul1 in alternative splicing and transcriptional regulation using RNA-sequencing, particularly of genes involved in translation, ubiquitination, and DNA damage. Given its cross-species conservation and role in splicing, it would not be surprising if it had a role in human development. Whole-exome sequencing detected a homozygous frameshift variant in HNRNPUL1 in 2 siblings with congenital limb malformations, which is a candidate gene for their limb malformations. Zebrafish Hnrnpul1 mutants suggest an important developmental role of hnRNPUL1 and provide motivation for exploring the potential conservation of ancient regulatory circuits involving hnRNPUL1 in human development.We thank the Alberta Children’s Hospital Research Institute for funding for DB and SF through the MORPH project. Operating funding was received from the Canadian Institute of Health Research Institute of Genetics Rare Disease Models and Mechanisms (180309-001-001) and from the National Science and Engineering Research Council (RGPIN/07176-2019) to SJ