Clinical exome sequencing data from patients with inborn errors of immunity: Cohort level diagnostic yield and the benefit of systematic reanalysis.

While next generation sequencing has expanded the scientific understanding of Inborn Errors of Immunity (IEI), the clinical use and re-use of exome sequencing is still emerging. We revisited clinical exome data from 1300 IEI patients using an updated in silico IEI gene panel. Variants were classified and curated through expert review. The molecular diagnostic yield after standard exome analysis was 11.8 %. Through systematic reanalysis, we identified variants of interest in 5.2 % of undiagnosed patients, with 76.7 % being (candidate) disease-causing, providing a (candidate) diagnosis in 15.2 % of our cohort. We find a 1.7 percentage point increase in conclusive molecular diagnoses. We find a high degree of actionability in patients with a genetic diagnosis (76.4 %). Despite the modest absolute diagnostic gain, these data support the benefit of iterative exome reanalysis in IEI patients, conveying the notion that our current understanding of genes and variants involved in IEI is by far not saturated.

Missense MED12 variants in 22 males with intellectual disability: From nonspecific symptoms to complete syndromes.

We describe the phenotype of 22 male patients (20 probands) carrying a hemizygous missense variant in MED12. The phenotypic spectrum is very broad ranging from nonspecific intellectual disability (ID) to the three well-known syndromes: Opitz-Kaveggia syndrome, Lujan-Fryns syndrome, or Ohdo syndrome. The identified variants were randomly distributed throughout the gene (p = 0.993, χ2 test), but mostly outside the functional domains (p = 0.004; χ2 test). Statistical analyses did not show a correlation between the MED12-related phenotypes and the locations of the variants (p = 0.295; Pearson correlation), nor the protein domain involved (p = 0.422; Pearson correlation). In conclusion, establishing a genotype-phenotype correlation in MED12-related diseases remains challenging. Therefore, we think that patients with a causative MED12 variant are currently underdiagnosed due to the broad patients' clinical presentations.

Phenotypic and genetic spectrum of ATP6V1A encephalopathy: a disorder of lysosomal homeostasis.

Vacuolar-type H+-ATPase (V-ATPase) is a multimeric complex present in a variety of cellular membranes that acts as an ATP-dependent proton pump and plays a key role in pH homeostasis and intracellular signalling pathways. In humans, 22 autosomal genes encode for a redundant set of subunits allowing the composition of diverse V-ATPase complexes with specific properties and expression. Sixteen subunits have been linked to human disease. Here we describe 26 patients harbouring 20 distinct pathogenic de novo missense ATP6V1A variants, mainly clustering within the ATP synthase α/ß family-nucleotide-binding domain. At a mean age of 7 years (extremes: 6 weeks, youngest deceased patient to 22 years, oldest patient) clinical pictures included early lethal encephalopathies with rapidly progressive massive brain atrophy, severe developmental epileptic encephalopathies and static intellectual disability with epilepsy. The first clinical manifestation was early hypotonia, in 70%; 81% developed epilepsy, manifested as developmental epileptic encephalopathies in 58% of the cohort and with infantile spasms in 62%; 63% of developmental epileptic encephalopathies failed to achieve any developmental, communicative or motor skills. Less severe outcomes were observed in 23% of patients who, at a mean age of 10 years and 6 months, exhibited moderate intellectual disability, with independent walking and variable epilepsy. None of the patients developed communicative language. Microcephaly (38%) and amelogenesis imperfecta/enamel dysplasia (42%) were additional clinical features. Brain MRI demonstrated hypomyelination and generalized atrophy in 68%. Atrophy was progressive in all eight individuals undergoing repeated MRIs. Fibroblasts of two patients with developmental epileptic encephalopathies showed decreased LAMP1 expression, Lysotracker staining and increased organelle pH, consistent with lysosomal impairment and loss of V-ATPase function. Fibroblasts of two patients with milder disease, exhibited a different phenotype with increased Lysotracker staining, decreased organelle pH and no significant modification in LAMP1 expression. Quantification of substrates for lysosomal enzymes in cellular extracts from four patients revealed discrete accumulation. Transmission electron microscopy of fibroblasts of four patients with variable severity and of induced pluripotent stem cell-derived neurons from two patients with developmental epileptic encephalopathies showed electron-dense inclusions, lipid droplets, osmiophilic material and lamellated membrane structures resembling phospholipids. Quantitative assessment in induced pluripotent stem cell-derived neurons identified significantly smaller lysosomes. ATP6V1A-related encephalopathy represents a new paradigm among lysosomal disorders. It results from a dysfunctional endo-lysosomal membrane protein causing altered pH homeostasis. Its pathophysiology implies intracellular accumulation of substrates whose composition remains unclear, and a combination of developmental brain abnormalities and neurodegenerative changes established during prenatal and early postanal development, whose severity is variably determined by specific pathogenic variants.

Cytosolic phosphoenolpyruvate carboxykinase deficiency: Expanding the clinical phenotype and novel laboratory findings.

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) deficiency due to the homozygous PCK1 variant has recently been associated with childhood-onset hypoglycemia with a recognizable pattern of abnormal urine organic acids. In this study, 21 children and 3 adult patients with genetically confirmed PEPCK-C deficiency were diagnosed during the years 2016 to 2019 and the available biochemical and clinical data were collected. All patients were ethnic Finns. Most patients (22 out of 24) had a previously published homozygous PCK1 variant c.925G>A. Two patients had a novel compound heterozygous PCK1 variant c.925G>A and c.716C>T. The laboratory results showed abnormal urine organic acid profile with increased tricarboxylic acid cycle intermediates and inadequate ketone body production during hypoglycemia. The hypoglycemic episodes manifested predominantly in the morning. Infections, fasting or poor food intake, heavy exercise, alcohol consumption, and breastfeeding were identified as triggering factors. Five patients presented with neonatal hypoglycemia. Hypoglycemic seizures occurred in half of the patients (12 out of 24). The first hypoglycemic episode often occurred at the age of 1-2 years, but it sometimes presented at a later age, and could re-occur during school age or adulthood. This study adds to the laboratory data on PEPCK-C deficiency, confirming the recognizable urine organic acid pattern and identifying deficient ketogenesis as a novel laboratory finding. The phenotype is expanded suggesting that the risk of hypoglycemia may continue into adulthood if predisposing factors are present.

Homozygous TAF1C variants are associated with a novel childhood-onset neurological phenotype.

TATA-box binding protein associated factor, RNA polymerase I subunit C (TAF1C) is a component of selectivity factor 1 belonging to RNA polymerase I (Pol I) transcription machinery. We report two unrelated patients with homozygous TAF1C missense variants and an early onset neurological phenotype with severe global developmental delay. Clinical features included lack of speech and ambulation and epilepsy. MRI of the brain demonstrated widespread cerebral atrophy and frontal periventricular white matter hyperintensity. The phenotype resembled that of a previously described variant of UBTF, which encodes another transcription factor of Pol I. TAF1C variants were located in two conserved amino acid positions and were predicted to be deleterious. In patient-derived fibroblasts, TAF1C mRNA and protein expression levels were substantially reduced compared with healthy controls. We propose that the variants impairing TAF1C expression are likely pathogenic and relate to a novel neurological disease. This study expands the disease spectrum related to Pol I transcription machinery, associating the TAF1C missense variants with a severe neurological phenotype for the first time.

Recessive MYH3 variants cause "Contractures, pterygia, and variable skeletal fusions syndrome 1B" mimicking Escobar variant multiple pterygium syndrome.

The multiple pterygium syndromes (MPS) are rare disorders with disease severity ranging from lethal to milder forms. The nonlethal Escobar variant MPS (EVMPS) is characterized by multiple pterygia and arthrogryposis, as well as various additional features including congenital anomalies. The genetic etiology of EVMPS is heterogeneous and the diagnosis has been based either on the detection of pathogenic CHRNG variants (~23% of patients), or suggestive clinical features. We describe four patients with a clinical suspicion of EVMPS who manifested with multiple pterygia, mild flexion contractures of several joints, and vertebral anomalies. We revealed recessively inherited MYH3 variants as the underlying cause in all patients: two novel variants, c.1053C>G, p.(Tyr351Ter) and c.3102+5G>C, as compound heterozygous with the hypomorphic MYH3 variant c.-9+1G>A. Recessive MYH3 variants have been previously associated with spondylocarpotarsal synostosis syndrome. Our findings now highlight multiple pterygia as an important feature in patients with recessive MYH3 variants. Based on all patients with recessive MYH3 variants reported up to date, we consider that this disease entity should be designated as "Contractures, pterygia, and variable skeletal fusions syndrome 1B," as recently suggested by OMIM. Our findings underline the importance of analyzing MYH3 in the differential diagnosis of EVMPS, particularly as the hypomorphic MYH3 variant might remain undetected by routine exome sequencing.

Biallelic Variants in UBA5 Link Dysfunctional UFM1 Ubiquitin-like Modifier Pathway to Severe Infantile-Onset Encephalopathy.

The ubiquitin fold modifier 1 (UFM1) cascade is a recently identified evolutionarily conserved ubiquitin-like modification system whose function and link to human disease have remained largely uncharacterized. By using exome sequencing in Finnish individuals with severe epileptic syndromes, we identified pathogenic compound heterozygous variants in UBA5, encoding an activating enzyme for UFM1, in two unrelated families. Two additional individuals with biallelic UBA5 variants were identified from the UK-based Deciphering Developmental Disorders study and one from the Northern Finland Intellectual Disability cohort. The affected individuals (n = 9) presented in early infancy with severe irritability, followed by dystonia and stagnation of development. Furthermore, the majority of individuals display postnatal microcephaly and epilepsy and develop spasticity. The affected individuals were compound heterozygous for a missense substitution, c.1111G>A (p.Ala371Thr; allele frequency of 0.28% in Europeans), and a nonsense variant or c.164G>A that encodes an amino acid substitution p.Arg55His, but also affects splicing by facilitating exon 2 skipping, thus also being in effect a loss-of-function allele. Using an in vitro thioester formation assay and cellular analyses, we show that the p.Ala371Thr variant is hypomorphic with attenuated ability to transfer the activated UFM1 to UFC1. Finally, we show that the CNS-specific knockout of Ufm1 in mice causes neonatal death accompanied by microcephaly and apoptosis in specific neurons, further suggesting that the UFM1 system is essential for CNS development and function. Taken together, our data imply that the combination of a hypomorphic p.Ala371Thr variant in trans with a loss-of-function allele in UBA5 underlies a severe infantile-onset encephalopathy.

Familial oligodontia and regional odontodysplasia associated with a PAX9 initiation codon mutation.

OBJECTIVE: Tooth agenesis is one of the most common craniofacial developmental anomalies. In hypodontia, one to five teeth are missing, whereas oligodontia refers to the absence of at least six teeth, excluding the third molars. Mutations in several genes including MSX1, PAX9, AXIN2, and WNT10A have been shown to cause non-syndromic tooth agenesis. Regional odontodysplasia (RO), also known as "ghost teeth," is a rare developmental anomaly of tooth formation affecting both dentitions. Some possible causes of RO have been suggested, yet the etiology remains unknown. Because the phenotypes of both oligodontia and RO co-occur in one Finnish family, the aim here was to investigate the genetic etiology of the two conditions. MATERIALS AND METHODS: A mutation screening of the genes MSX1, PAX9, AXIN2, and WNT10A was performed for the family members of a RO patient and family history of oligodontia. RESULTS: An initiation codon mutation of the PAX9 gene was found in the proband and segregating with oligodontia in the family. CONCLUSIONS: The etiology of regional odontodysplasia (RO) may be genetic and the same genes can be involved both in RO and tooth agenesis. CLINICAL RELEVANCE: Our results give new insights into the etiology of regional odontodysplasia, yet further results are needed.

Novel homozygous PCK1 mutation causing cytosolic phosphoenolpyruvate carboxykinase deficiency presenting as childhood hypoglycemia, an abnormal pattern of urine metabolites and liver dysfunction.

Clinical and laboratory data were collected from three Finnish patients including a sibling pair and another unrelated child with unexplained childhood hypoglycemia. Transient elevation of alanine transaminase, lactate and tricarboxylic acid cycle intermediates, especially fumarate, were noticed in urine organic acid analysis. Exome sequencing was performed for the patients and their parents. A novel homozygous PCK1 c.925G>A (p.G309R) mutation was detected in all affected individuals. COS-1 cells transfected with mutant PCK1 transcripts were used to study the pathogenic nature of the detected variant. The COS-1 transfected cells showed the mutant gene to be incapable of producing a normally functioning cytosolic phosphoenolpyruvate carboxykinase (PEPCK) enzyme. This report further delineates the clinical phenotype of isolated cytosolic PEPCK deficiency and offers a metabolic pattern helping to recognize these patients. Cytosolic PEPCK deficiency should be considered in the differential diagnosis of children presenting with hypoglycemia, hepatic dysfunction and elevated tricarboxylic acid intermediates in urinary organic acid analysis.

Riboflavin-Responsive Multiple Acyl-CoA Dehydrogenase Deficiency Associated with Hepatoencephalomyopathy and White Matter Signal Abnormalities on Brain MRI.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare inborn error of metabolism affecting both fatty acid and amino acid oxidation. It can manifest at any age, but riboflavin-responsiveness has mainly been described in less severely affected patients. We describe an infant with severe MADD presenting with profound hypotonia and hepatomegaly. Treatment with riboflavin improved his muscle strength, liver size, and biochemical markers. A homozygous mutation of electron transfer flavoprotein dehydrogenase (ETFDH) was found. His motor skills continued to progress until a fatal infection-triggered deterioration at the age of 34 months. We show changes in brain magnetic resonance imaging over the course of the disease, with profound white matter abnormalities during the deterioration phase. Aggregates of mitochondria with abnormal cristae in muscle electron microscopy were noticed already in infancy. An unusual lactate dehydrogenase (LDH) isoenzyme pattern with LDH-1 predominance was additionally observed. This case demonstrates riboflavin-responsiveness in a severely affected infant with both muscular and extramuscular involvement and further underlines the variable nature of this disease.

Adrenomyeloneuropathy due to mutation in the ABCD1 gene as underlying factor in spastic paraparesis.

We present a Finnish family in which adrenomyeloneuropathy (AMN) caused by the mutation in the ABCD1 gene was revealed as the cause of spastic paraparesis. . Two patients had hypoadrenalism, which is in some cases some associated with the disease . AMN is a hereditary disease manifested both in men and women. but owing to the location of the gene in the X chromosome the symptoms are usually more severe in male patients. . Diagnoses was trucked down with gene-panel sequencing and confirmed through detection of an elevated level of very long-chain fatty acids in the serum of the patients. Specific molecular genetic diagnosis is beneficial, because it enables precise genetic counseling as well as recognition and treatment of associated symptoms, such as severe cortisol deficiency.

Evidence for the additivity of rare and common variant burden throughout the spectrum of intellectual disability.

Intellectual disability (ID) is a common disorder, yet there is a wide spectrum of impairment from mild to profoundly affected individuals. Mild ID is seen as the low extreme of the general distribution of intelligence, while severe ID is often seen as a monogenic disorder caused by rare, pathogenic, highly penetrant variants. To investigate the genetic factors influencing mild and severe ID, we evaluated rare and common variation in the Northern Finland Intellectual Disability cohort (n = 1096 ID patients), a cohort with a high percentage of mild ID (n = 550) and from a population bottleneck enriched in rare, damaging variation. Despite this enrichment, we found only a small percentage of ID was due to recessive Finnish-enriched variants (0.5%). A larger proportion was linked to dominant variation, with a significant burden of rare, damaging variation in both mild and severe ID. This rare variant burden was enriched in more severe ID (p = 2.4e-4), patients without a relative with ID (p = 4.76e-4), and in those with features associated with monogenic disorders. We also found a significant burden of common variants associated with decreased cognitive function, with no difference between mild and more severe ID. When we included common and rare variants in a joint model, the rare and common variants had additive effects in both mild and severe ID. A multimodel inference approach also found that common and rare variants together best explained ID status (ΔAIC = 16.8, ΔBIC = 10.2). Overall, we report evidence for the additivity of rare and common variant burden throughout the spectrum of intellectual disability.

Clinical and Genetic Characteristics of Finnish Patients with Autosomal Recessive and Dominant Non-Syndromic Hearing Loss Due to Pathogenic TMC1 Variants.

Sensorineural hearing loss (SNHL) is one of the most common sensory deficits worldwide, and genetic factors contribute to at least 50−60% of the congenital hearing loss cases. The transmembrane channel-like protein 1 (TMC1) gene has been linked to autosomal recessive (DFNB7/11) and autosomal dominant (DFNA36) non-syndromic hearing loss, and it is a relatively common genetic cause of SNHL. Here, we report eight Finnish families with 11 affected family members with either recessively inherited homozygous or compound heterozygous TMC1 variants associated with congenital moderate-to-profound hearing loss, or a dominantly inherited heterozygous TMC1 variant associated with postlingual progressive hearing loss. We show that the TMC1 c.1534C>T, p.(Arg512*) variant is likely a founder variant that is enriched in the Finnish population. We describe a novel recessive disease-causing TMC1 c.968A>G, p.(Tyr323Cys) variant. We also show that individuals in this cohort who were diagnosed early and received timely hearing rehabilitation with hearing aids and cochlear implants (CI) have reached good speech perception in noise. Comparison of the genetic data with the outcome of CI rehabilitation increases our understanding of the extent to which underlying pathogenic gene variants explain the differences in CI rehabilitation outcomes.

Heterozygous premature termination in zinc-finger domain of Krüppel-like factor 2 gene associates with dysregulated immunity.

Krüppel-like factor 2 (KLF2) is a transcription factor with significant roles in development, maturation, differentiation, and proliferation of several cell types. In immune cells, KLF2 regulates maturation and trafficking of lymphocytes and monocytes. KLF2 participates in regulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Although pulmonary arterial hypertension (PAH) related to KLF2 genetic variant has been suggested, genetic role of KLF2 associated with immune dysregulation has not been described. We identified a family whose members suffered from lymphopenia, autoimmunity, and malignancy. Whole exome sequencing revealed a KLF2 p.(Glu318Argfs*87) mutation disrupting the highly conserved zinc finger domain. We show a reduced amount of KLF2 protein, defective nuclear localization and altered protein-protein interactome. The phenotypically variable positive cases presented with B and T cell lymphopenia and abnormalities in B and T cell maturation including low naive T cell counts and low CD27+IgD-IgM- switched memory B cells. KLF2 target gene (CD62L) expression was affected. Although the percentage of (CD25+FOXP3+, CD25+CD127-) regulatory T cells (Treg) was high, the naive Treg cells (CD45RA+) were absent. Serum IgG1 levels were low and findings in one case were consistent with common variable immunodeficiency (CVID). Transcription of NF-κß pathway genes and p65/RelA phosphorylation were not significantly affected. Inflammasome activity, transcription of genes related with JAK/STAT pathway and interferon signature were also comparable to controls. Evidence of PAH was not found. In conclusion, KLF2 variant may be associated with familial immune dysregulation. Although the KLF2 deficient family members in our study suffered from lymphopenia, autoimmunity or malignancy, additional study cohorts are required to confirm our observations.

Collagen XIII-derived ectodomain regulates bone angiogenesis and intracortical remodeling.

Osteoporosis is the most common degenerative bone disease that occurs when the balance of bone production and resorption is perturbed. Loss of bone mass or alteration in its quality leads to significant weakening of the bones and subsequently to higher fracture risk. Collagen XIII (ColXIII) is a conserved transmembrane protein expressed in many mesenchymal tissues. Here we show that ColXIII is a regulator of bone remodeling niche. In this study, we found that ColXIII expression is significantly upregulated in osteoporotic patients. In view of that, we studied bone homeostasis in ColXIII-overexpressing mice (Col13a1oe) up to 72 weeks of age and observed a cortical bone overgrowth followed by a drastic bone loss, together with increased bone vascularization. Moreover, our results demonstrate that the ColXIII-derived ectodomain enhances angiogenesis through ß1-integrins and the JNK pathway. Consequently, these data suggest that ColXIII has a role in age-dependent cortical bone deterioration with possible implications for osteoporosis and fracture risk.

Contribution of rare and common variants to intellectual disability in a sub-isolate of Northern Finland.

The contribution of de novo variants in severe intellectual disability (ID) has been extensively studied whereas the genetics of mild ID has been less characterized. To elucidate the genetics of milder ID we studied 442 ID patients enriched for mild ID (>50%) from a population isolate of Finland. Using exome sequencing, we show that rare damaging variants in known ID genes are observed significantly more often in severe (27%) than in mild ID (13%) patients. We further observe a significant enrichment of functional variants in genes not yet associated with ID (OR: 2.1). We show that a common variant polygenic risk significantly contributes to ID. The heritability explained by polygenic risk score is the highest for educational attainment (EDU) in mild ID (2.2%) but lower for more severe ID (0.6%). Finally, we identify a Finland enriched homozygote variant in the CRADD ID associated gene.

Exome sequencing in routine diagnostics: a generic test for 254 patients with primary immunodeficiencies.

BACKGROUND: Diagnosis of primary immunodeficiencies (PIDs) is complex and cumbersome yet important for the clinical management of the disease. Exome sequencing may provide a genetic diagnosis in a significant number of patients in a single genetic test. METHODS: In May 2013, we implemented exome sequencing in routine diagnostics for patients suffering from PIDs. This study reports the clinical utility and diagnostic yield for a heterogeneous group of 254 consecutively referred PID patients from 249 families. For the majority of patients, the clinical diagnosis was based on clinical criteria including rare and/or unusual severe bacterial, viral, or fungal infections, sometimes accompanied by autoimmune manifestations. Functional immune defects were interpreted in the context of aberrant immune cell populations, aberrant antibody levels, or combinations of these factors. RESULTS: For 62 patients (24%), exome sequencing identified pathogenic variants in well-established PID genes. An exome-wide analysis diagnosed 10 additional patients (4%), providing diagnoses for 72 patients (28%) from 68 families altogether. The genetic diagnosis directly indicated novel treatment options for 25 patients that received a diagnosis (34%). CONCLUSION: Exome sequencing as a first-tier test for PIDs granted a diagnosis for 28% of patients. Importantly, molecularly defined diagnoses indicated altered therapeutic options in 34% of cases. In addition, exome sequencing harbors advantages over gene panels as a truly generic test for all genetic diseases, including in silico extension of existing gene lists and re-analysis of existing data.

Two missense mutations in KCNQ1 cause pituitary hormone deficiency and maternally inherited gingival fibromatosis.

Familial growth hormone deficiency provides an opportunity to identify new genetic causes of short stature. Here we combine linkage analysis with whole-genome resequencing in patients with growth hormone deficiency and maternally inherited gingival fibromatosis. We report that patients from three unrelated families harbor either of two missense mutations, c.347G>T p.(Arg116Leu) or c.1106C>T p.(Pro369Leu), in KCNQ1, a gene previously implicated in the long QT interval syndrome. Kcnq1 is expressed in hypothalamic GHRH neurons and pituitary somatotropes. Co-expressing KCNQ1 with the KCNE2 ß-subunit shows that both KCNQ1 mutants increase current levels in patch clamp analyses and are associated with reduced pituitary hormone secretion from AtT-20 cells. In conclusion, our results reveal a role for the KCNQ1 potassium channel in the regulation of human growth, and show that growth hormone deficiency associated with maternally inherited gingival fibromatosis is an allelic disorder with cardiac arrhythmia syndromes caused by KCNQ1 mutations.