Creation of a structured molecular genomics report for Germany as a local adaption of HL7's Genomic Reporting Implementation Guide.

OBJECTIVE: The objective was to develop a dataset definition, information model, and FHIR® specification for key data elements contained in a German molecular genomics (MolGen) report to facilitate genomic and phenotype integration in electronic health records. MATERIALS AND METHODS: A dedicated expert group participating in the German Medical Informatics Initiative reviewed information contained in MolGen reports, determined the key elements, and formulated a dataset definition. HL7's Genomics Reporting Implementation Guide (IG) was adopted as a basis for the FHIR® specification which was subjected to a public ballot. In addition, elements in the MolGen dataset were mapped to the fields defined in ISO/TS 20428:2017 standard to evaluate compliance. RESULTS: A core dataset of 76 data elements, clustered into 6 categories was created to represent all key information of German MolGen reports. Based on this, a FHIR specification with 16 profiles, 14 derived from HL7®'s Genomics Reporting IG and 2 additional profiles (of the FamilyMemberHistory and RiskAssessment resources), was developed. Five example resource bundles show how our adaptation of an international standard can be used to model MolGen report data that was requested following oncological or rare disease indications. Furthermore, the map of the MolGen report data elements to the fields defined by the ISO/TC 20428:2017 standard, confirmed the presence of the majority of required fields. CONCLUSIONS: Our report serves as a template for other research initiatives attempting to create a standard format for unstructured genomic report data. Use of standard formats facilitates integration of genomic data into electronic health records for clinical decision support.

An intronic splice site alteration in combination with a large deletion affecting VPS13B (COH1) causes Cohen syndrome.

Cohen syndrome (CS) is a rare, autosomal recessive disorder characterized by intellectual disability, postnatal microcephaly, facial abnormalities, abnormal truncal fat distribution, myopia, and pigmentary retinopathy. It is often considered an underdiagnosed condition, especially in children with developmental delay and intellectual disability. Here we report on four individuals from a large Jordanian family clinically diagnosed with CS. Using Trio Exome Sequencing (Trio-WES) and MLPA analyses we identified a maternally inherited novel intronic nucleotide substitution c.3446-23T>G leading to the activation of a cryptic splice site and a paternally inherited multi-exon deletion in VPS13B (previously termed COH1) in the index patient. Expression analysis showed a strong decrease of VPS13B mRNA levels and direct sequencing of cDNA confirmed splicing at a cryptic upstream splice acceptor site, resulting in the inclusion of 22 intronic bases. This extension results in a frameshift and a premature stop of translation (p.Gly1149Valfs*9). Segregation analysis revealed that three affected maternal cousins were homozygous for the intronic splice site variant. Our data show causality of both alterations and strongly suggest the expansion of the diagnostic strategy to search for intronic splice variants in molecularly unconfirmed patients affected by CS.

Just Expect It: Compound Heterozygous Variants of POMT1 in a Consanguineous Family-The Role of Next Generation Sequencing in Neuromuscular Disorders.

Muscular dystrophy-dystroglycanopathies (MDDG) are a group of genetically heterogeneous autosomal recessive disorders characterized by hypoglycosylation of α-dystroglycan. Here, we report on two female patients from a consanguineous Lebanese family that presented in early infancy with generalized muscle hypotonia and primary microcephaly. Brain magnetic resonance imaging (MRI) showed different degrees of hypoplasia of the cerebellar vermis and hypoplasia of corpus callosum. Muscle biopsy analyses revealed a muscular dystrophy with reduced expression of α-dystroglycan and merosin in immunoblot analyses. Homozygosity mapping failed to elucidate the causal mutation due to the accepted notion that, in consanguineous families, homozygote mutations cause disease. However, by applying whole exome sequencing, we identified a novel compound heterozygous POMT1 mutation that segregates with the phenotype and is in line with the clinical presentation. This underscores that a less expected compound heterozygous instead of homozygous mutation in a consanguineous marriage results in a recessive disorder and highlights the growing role of next generation sequencing in neuromuscular disorder diagnostics.

VarWatch-A stand-alone software tool for variant matching.

Massively parallel DNA sequencing of clinical samples holds great promise for the gene-based diagnosis of human inherited diseases because it allows rapid detection of putatively causative mutations at genome-wide level. Without additional evidence complementing their initial bioinformatics evaluation, however, the clinical relevance of such candidate genetic variants often remains unclear. In consequence, dedicated 'matching' services have been established in recent years that aim at the discovery of other, comparable case reports to facilitate individual diagnoses. However, legal concerns have been raised about the global sharing of genetic data, particularly in Europe where the recently enacted General Data Protection Regulation EU-2016/679 classifies genetic data as highly sensitive. Hence, unrestricted sharing of genetic data from clinical cases on platforms outside the national jurisdiction increasingly may be perceived as problematic. To allow collaborative data producers, particularly large consortia of diagnostic laboratories, to acknowledge these concerns while still practicing efficient case matching internally, novel tools are required. To this end, we developed VarWatch, an easy-to-deploy and highly scalable case matching software that provides users with comprehensive programmatic tools and a user-friendly interface to fulfil said purpose.

Genetics of intellectual disability in consanguineous families.

Autosomal recessive (AR) gene defects are the leading genetic cause of intellectual disability (ID) in countries with frequent parental consanguinity, which account for about 1/7th of the world population. Yet, compared to autosomal dominant de novo mutations, which are the predominant cause of ID in Western countries, the identification of AR-ID genes has lagged behind. Here, we report on whole exome and whole genome sequencing in 404 consanguineous predominantly Iranian families with two or more affected offspring. In 219 of these, we found likely causative variants, involving 77 known and 77 novel AR-ID (candidate) genes, 21 X-linked genes, as well as 9 genes previously implicated in diseases other than ID. This study, the largest of its kind published to date, illustrates that high-throughput DNA sequencing in consanguineous families is a superior strategy for elucidating the thousands of hitherto unknown gene defects underlying AR-ID, and it sheds light on their prevalence.

Effect of inbreeding on intellectual disability revisited by trio sequencing.

In outbred Western populations, most individuals with intellectual disability (ID) are sporadic cases, dominant de novo mutations (DNM) are frequent, and autosomal recessive ID (ARID) is very rare. Because of the high rate of parental consanguinity, which raises the risk for ARID and other recessive disorders, the prevalence of ID is significantly higher in near- and middle-east countries. Indeed, homozygosity mapping and sequencing in consanguineous families have already identified a plethora of ARID genes, but because of the design of these studies, DNMs could not be systematically assessed, and the proportion of cases that are potentially preventable by avoiding consanguineous marriages or through carrier testing is hitherto unknown. This prompted us to perform whole-exome sequencing in 100 sporadic ID patients from Iran and their healthy consanguineous parents. In 61 patients, we identified apparently causative changes in known ID genes. Of these, 44 were homozygous recessive and 17 dominant DNMs. Assuming that the DNM rate is stable, these results suggest that parental consanguinity raises the ID risk about 3.6-fold, and about 4.1 to 4.25-fold for children of first-cousin unions. These results do not rhyme with recent opinions that consanguinity-related health risks are generally small and have been "overstated" in the past.

Biallelic missense variants in ZBTB11 can cause intellectual disability in humans.

Exploring genes and pathways underlying intellectual disability (ID) provides insight into brain development and function, clarifying the complex puzzle of how cognition develops. As part of ongoing systematic studies to identify candidate ID genes, linkage analysis and next-generation sequencing revealed Zinc Finger and BTB Domain Containing 11 (ZBTB11) as a novel candidate ID gene. ZBTB11 encodes a little-studied transcription regulator, and the two identified missense variants in this study are predicted to disrupt canonical Zn2+-binding residues of its C2H2 zinc finger domain, leading to possible altered DNA binding. Using HEK293T cells transfected with wild-type and mutant GFP-ZBTB11 constructs, we found the ZBTB11 mutants being excluded from the nucleolus, where the wild-type recombinant protein is predominantly localized. Pathway analysis applied to ChIP-seq data deposited in the ENCODE database supports the localization of ZBTB11 in nucleoli, highlighting associated pathways such as ribosomal RNA synthesis, ribosomal assembly, RNA modification and stress sensing, and provides a direct link between subcellular ZBTB11 location and its function. Furthermore, given the report of prominent brain and spinal cord degeneration in a zebrafish Zbtb11 mutant, we investigated ZBTB11-ortholog knockdown in Drosophila melanogaster brain by targeting RNAi using the UAS/Gal4 system. The observed approximate reduction to a third of the mushroom body size-possibly through neuronal reduction or degeneration-may affect neuronal circuits in the brain that are required for adaptive behavior, specifying the role of this gene in the nervous system. In conclusion, we report two ID families segregating ZBTB11 biallelic mutations disrupting Zn2+-binding motifs and provide functional evidence linking ZBTB11 dysfunction to this phenotype.

Homozygous ARHGEF2 mutation causes intellectual disability and midbrain-hindbrain malformation.

Mid-hindbrain malformations can occur during embryogenesis through a disturbance of transient and localized gene expression patterns within these distinct brain structures. Rho guanine nucleotide exchange factor (ARHGEF) family members are key for controlling the spatiotemporal activation of Rho GTPase, to modulate cytoskeleton dynamics, cell division, and cell migration. We identified, by means of whole exome sequencing, a homozygous frameshift mutation in the ARHGEF2 as a cause of intellectual disability, a midbrain-hindbrain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turkish descent. We show that loss of ARHGEF2 perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. The ARHGEF2 mutation leads to reduction in the activation of the RhoA/ROCK/MLC pathway crucial for cell migration. We demonstrate that the human brain malformation is recapitulated in Arhgef2 mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the midbrain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation in ARHGEF2 as novel cause of a neurodevelopmental disorder.

Mutations of the aminoacyl-tRNA-synthetases SARS and WARS2 are implicated in the etiology of autosomal recessive intellectual disability.

Intellectual disability (ID) is the hallmark of an extremely heterogeneous group of disorders that comprises a wide variety of syndromic and non-syndromic phenotypes. Here, we report on mutations in two aminoacyl-tRNA synthetases that are associated with ID in two unrelated Iranian families. In the first family, we identified a homozygous missense mutation (c.514G>A, p.Asp172Asn) in the cytoplasmic seryl-tRNA synthetase (SARS) gene. The mutation affects the enzymatic core domain of the protein and impairs its enzymatic activity, probably leading to reduced cytoplasmic tRNASer concentrations. The mutant protein was predicted to be unstable, which could be substantiated by investigating ectopic mutant SARS in transfected HEK293T cells. In the second family, we found a compound heterozygous genotype of the mitochondrial tryptophanyl-tRNA synthetase (WARS2) gene, comprising a nonsense mutation (c.325delA, p.Ser109Alafs*15), which very likely entails nonsense-mediated mRNA decay and a missense mutation (c.37T>G, p.Trp13Gly). The latter affects the mitochondrial localization signal of WARS2, causing protein mislocalization. Including AIMP1, which we have recently implicated in the etiology of ID, three genes with a role in tRNA-aminoacylation are now associated with this condition. We therefore suggest that the functional integrity of tRNAs in general is an important factor in the development and maintenance of human cognitive functions.

Klüver-Bucy syndrome associated with a recessive variant in HGSNAT in two siblings with Mucopolysaccharidosis type IIIC (Sanfilippo C).

Klüver-Bucy syndrome (KBS) comprises a set of neurobehavioral symptoms with psychic blindness, hypersexuality, disinhibition, hyperorality, and hypermetamorphosis that were originally observed after bilateral lobectomy in Rhesus monkeys. We investigated two siblings with KBS from a consanguineous family by whole-exome sequencing and autozygosity mapping. We detected a homozygous variant in the heparan-α-glucosaminidase-N-acetyltransferase gene (HGSNAT; c.518G>A, p.(G173D), NCBI ClinVar RCV000239404.1), which segregated with the phenotype. Disease-causing variants in this gene are known to be associated with autosomal recessive Mucopolysaccharidosis type IIIC (MPSIIIC, Sanfilippo C). This lysosomal storage disease is due to deficiency of the acetyl-CoA:α-glucosaminidase-N-acetyltransferase, which was shown to be reduced in patient fibroblasts. Our report extends the phenotype associated with MPSIIIC. Besides MPSIIIA and MPSIIIB, due to variants in SGSH and NAGLU, this is the third subtype of Sanfilippo disease to be associated with KBS. MPSIII should be included in the differential diagnosis of young patients with KBS.

Homozygous YME1L1 mutation causes mitochondriopathy with optic atrophy and mitochondrial network fragmentation.

Mitochondriopathies often present clinically as multisystemic disorders of primarily high-energy consuming organs. Assembly, turnover, and surveillance of mitochondrial proteins are essential for mitochondrial function and a key task of AAA family members of metalloproteases. We identified a homozygous mutation in the nuclear encoded mitochondrial escape 1-like 1 gene YME1L1, member of the AAA protease family, as a cause of a novel mitochondriopathy in a consanguineous pedigree of Saudi Arabian descent. The homozygous missense mutation, located in a highly conserved region in the mitochondrial pre-sequence, inhibits cleavage of YME1L1 by the mitochondrial processing peptidase, which culminates in the rapid degradation of YME1L1 precursor protein. Impaired YME1L1 function causes a proliferation defect and mitochondrial network fragmentation due to abnormal processing of OPA1. Our results identify mutations in YME1L1 as a cause of a mitochondriopathy with optic nerve atrophy highlighting the importance of YME1L1 for mitochondrial functionality in humans.

Replication of the association between CHRNA4 rs1044396 and harm avoidance in a large population-based sample.

Harm avoidance is a personality trait characterized by excessive worrying and fear of uncertainty, which has repeatedly been related to anxiety disorders. Converging lines of research in rodents and humans point towards an involvement of the nicotinic cholinergic system in the modulation of anxiety. Most notably, the rs1044396 polymorphism in the CHRNA4 gene, which codes for the α4 subunit of the nicotinic acetylcholine receptor, has been linked to negative emotionality traits including harm avoidance in a recent study. Against this background, we investigated the association between harm avoidance and the rs1044396 polymorphism using data from N=1673 healthy subjects, which were collected in the context of the German multi-centre study ׳Genetics of Nicotine Dependence and Neurobiological Phenotypes׳. Homozygous carriers of the C-allele showed significantly higher levels of harm avoidance than homozygous T-allele carriers, with heterozygous subjects exhibiting intermediate scores. The effect was neither modulated by age or gender nor by smoking status. By replicating previous findings in a large population-based sample for the first time, the present study adds to the growing evidence suggesting an involvement of nicotinic cholinergic mechanism in anxiety and negative emotionality, which may pose an effective target for medical treatment.

Missense variants in AIMP1 gene are implicated in autosomal recessive intellectual disability without neurodegeneration.

AIMP1/p43 is a multifunctional non-catalytic component of the multisynthetase complex. The complex consists of nine catalytic and three non-catalytic proteins, which catalyze the ligation of amino acids to their cognate tRNA isoacceptors for use in protein translation. To date, two allelic variants in the AIMP1 gene have been reported as the underlying cause of autosomal recessive primary neurodegenerative disorder. Here, we present two consanguineous families from Pakistan and Iran, presenting with moderate to severe intellectual disability, global developmental delay, and speech impairment without neurodegeneration. By the combination of homozygosity mapping and next generation sequencing, we identified two homozygous missense variants, p.(Gly299Arg) and p.(Val176Gly), in the gene AIMP1 that co-segregated with the phenotype in the respective families. Molecular modeling of the variants revealed deleterious effects on the protein structure that are predicted to result in reduced AIMP1 function. Our findings indicate that the clinical spectrum for AIMP1 defects is broader than witnessed so far.

[From phenotype to genotype: a glimpse behind the scenes of an unending story]. / Die Reise vom Phänotyp zum Genotyp.

Mendelian conditions direct attention at basic mechanisms. In the 1990's DNA sequencing allowed elucidating such conditions. We embarked on an unexpected adventure to study a monogenic autosomal-dominant form of hypertension causing also a specific form of short fingers. The gene locus caused a 50 mmHg increase in blood pressure at age of 50. Our clinically based group stumbled to the finish line after 20 years of study. We remained together and proudly persevered. Our findings could be relevant for essential hypertension.

Penetrance of pathogenic mutations in haploinsufficient genes for intellectual disability and related disorders.

De novo loss of function (LOF) mutations in the ASXL3 gene cause Bainbridge-Ropers syndrome, a severe form of intellectual disability (ID) and developmental delay, but there is evidence that they also occur in healthy individuals. This has prompted us to look for non-pathogenic LOF variants in other ID genes. Heterozygous LOF mutations in ASXL1, a paralog of ASXL3, are known to cause Bohring-Opitz syndrome (BOS), and benign LOF mutations in this gene have not been published to date. Therefore, we were surprised to find 56 ASXL1 LOF variants in the ExAC database (http://exac.broadinstitute.org), comprising exomes from 60,706 individuals who had been selected to exclude severe genetic childhood disorders. 4 of these variants have been described as disease-causing in patients with BOS, which rules out the possibility that pathogenic and clinically neutral LOF variants in this gene are functionally distinct. Apparently benign LOF variants were also detected in several other genes for ID and related disorders, including CDH15, KATNAL2, DEPDC5, ARID1B and AUTS2, both in the ExAC database and in the 6,500 exomes of the Exome Variant Server (http://evs.gs.washington.edu/EVS/). These observations argue for low penetrance of LOF mutations in ASXL1 and other genes for ID and related disorders, which could have far-reaching implications for genetic counseling and research.

Exome Sequencing and Linkage Analysis Identified Novel Candidate Genes in Recessive Intellectual Disability Associated with Ataxia.

BACKGROUND: Intellectual disability (ID) is a neuro-developmental disorder which causes considerable socio-economic problems. Some ID individuals are also affected by ataxia, and the condition includes different mutations affecting several genes. METHODS: We used whole exome sequencing (WES) in combination with homozygosity mapping (HM) to identify the genetic defects in five consanguineous families among our cohort study, with two affected children with ID and ataxia as major clinical symptoms. RESULTS: We identified three novel candidate genes, RIPPLY1, MRPL10, SNX14, and a new mutation in known gene SURF1. All are autosomal genes, except RIPPLY1, which is located on the X chromosome. Two are housekeeping genes, implicated in transcription and translation regulation and intracellular trafficking, and two encode mitochondrial proteins. The pathogenesis of these variants was evaluated by mutation classification, bioinformatic methods, review of medical and biological relevance, co-segregation studies in the particular family, and a normal population study. CONCLUSIONS: Linkage analysis and exome sequencing of a small number of affected family members is a powerful new technique which can be used to decrease the number of candidate genes in heterogenic disorders such as ID, and may even identify the responsible gene(s).

The Role of a Novel TRMT1 Gene Mutation and Rare GRM1 Gene Defect in Intellectual Disability in Two Azeri Families.

Cognitive impairment or intellectual disability (ID) is a widespread neurodevelopmental disorder characterized by low IQ (below 70). ID is genetically heterogeneous and is estimated to affect 1-3% of the world's population. In affected children from consanguineous families, autosomal recessive inheritance is common, and identifying the underlying genetic cause is an important issue in clinical genetics. In the framework of a larger project, aimed at identifying candidate genes for autosomal recessive intellectual disorder (ARID), we recently carried out single nucleotide polymorphism-based genome-wide linkage analysis in several families from Ardabil province in Iran. The identification of homozygosity-by-descent loci in these families, in combination with whole exome sequencing, led us to identify possible causative homozygous changes in two families. In the first family, a missense variant was found in GRM1 gene, while in the second family, a frameshift alteration was identified in TRMT1, both of which were found to co-segregate with the disease. GRM1, a known causal gene for autosomal recessive spinocerebellar ataxia (SCAR13, MIM#614831), encodes the metabotropic glutamate receptor1 (mGluR1). This gene plays an important role in synaptic plasticity and cerebellar development. Conversely, the TRMT1 gene encodes a tRNA methyltransferase that dimethylates a single guanine residue at position 26 of most tRNAs using S-adenosyl methionine as the methyl group donor. We recently presented TRMT1 as a candidate gene for ARID in a consanguineous Iranian family (Najmabadi et al., 2011). We believe that this second Iranian family with a biallelic loss-of-function mutation in TRMT1 gene supports the idea that this gene likely has function in development of the disorder.

Evaluating information content of SNPs for sample-tagging in re-sequencing projects.

Sample-tagging is designed for identification of accidental sample mix-up, which is a major issue in re-sequencing studies. In this work, we develop a model to measure the information content of SNPs, so that we can optimize a panel of SNPs that approach the maximal information for discrimination. The analysis shows that as low as 60 optimized SNPs can differentiate the individuals in a population as large as the present world, and only 30 optimized SNPs are in practice sufficient in labeling up to 100 thousand individuals. In the simulated populations of 100 thousand individuals, the average Hamming distances, generated by the optimized set of 30 SNPs are larger than 18, and the duality frequency, is lower than 1 in 10 thousand. This strategy of sample discrimination is proved robust in large sample size and different datasets. The optimized sets of SNPs are designed for Whole Exome Sequencing, and a program is provided for SNP selection, allowing for customized SNP numbers and interested genes. The sample-tagging plan based on this framework will improve re-sequencing projects in terms of reliability and cost-effectiveness.

PDE3A mutations cause autosomal dominant hypertension with brachydactyly.

Cardiovascular disease is the most common cause of death worldwide, and hypertension is the major risk factor. Mendelian hypertension elucidates mechanisms of blood pressure regulation. Here we report six missense mutations in PDE3A (encoding phosphodiesterase 3A) in six unrelated families with mendelian hypertension and brachydactyly type E (HTNB). The syndrome features brachydactyly type E (BDE), severe salt-independent but age-dependent hypertension, an increased fibroblast growth rate, neurovascular contact at the rostral-ventrolateral medulla, altered baroreflex blood pressure regulation and death from stroke before age 50 years when untreated. In vitro analyses of mesenchymal stem cell-derived vascular smooth muscle cells (VSMCs) and chondrocytes provided insights into molecular pathogenesis. The mutations increased protein kinase A-mediated PDE3A phosphorylation and resulted in gain of function, with increased cAMP-hydrolytic activity and enhanced cell proliferation. Levels of phosphorylated VASP were diminished, and PTHrP levels were dysregulated. We suggest that the identified PDE3A mutations cause the syndrome. VSMC-expressed PDE3A deserves scrutiny as a therapeutic target for the treatment of hypertension.

New evidence for the role of calpain 10 in autosomal recessive intellectual disability: identification of two novel nonsense variants by exome sequencing in Iranian families.

BACKGROUND: Knowledge of the genes responsible for intellectual disability, particularly autosomal recessive forms, is rapidly expanding. Increasing numbers of the gene show great heterogeneity and supports the hypothesis that human genome may contain over 2000 causative genes with a critical role in brain development. METHODS: Since 2004, we have applied genome-wide SNP genotyping and next-generation sequencing in large consanguineous Iranian families with intellectual disability, to identify the genes harboring disease-causing mutations. The current study paved the way for identification of responsible genes in two unrelated Iranian families. RESULTS: We found two novel nonsense mutations, p.C77* and p.Q115*, in the calpain catalytic domain of CAPN10, which is a cysteine protease known to be involved in pathogenesis of noninsulin-dependent diabetes mellitus. Another different mutation in this gene (p.S138_R139ins5) has previously been reported in an Iranian family. All of these patients have common clinical features in spite of specific brain structural abnormalities on MRI. CONCLUSIONS: Different mutations in CAPN10 have already been found in three independent Iranian families. These results have strongly supported the possible role of CAPN10 in human brain development. Altogether, we proposed CAPN10 as a promising candidate gene for intellectual disability, which should be considered in diagnostic gene panels.