Chromosome catastrophes involve replication mechanisms generating complex genomic rearrangements.

Complex genomic rearrangements (CGRs) consisting of two or more breakpoint junctions have been observed in genomic disorders. Recently, a chromosome catastrophe phenomenon termed chromothripsis, in which numerous genomic rearrangements are apparently acquired in one single catastrophic event, was described in multiple cancers. Here, we show that constitutionally acquired CGRs share similarities with cancer chromothripsis. In the 17 CGR cases investigated, we observed localization and multiple copy number changes including deletions, duplications, and/or triplications, as well as extensive translocations and inversions. Genomic rearrangements involved varied in size and complexities; in one case, array comparative genomic hybridization revealed 18 copy number changes. Breakpoint sequencing identified characteristic features, including small templated insertions at breakpoints and microhomology at breakpoint junctions, which have been attributed to replicative processes. The resemblance between CGR and chromothripsis suggests similar mechanistic underpinnings. Such chromosome catastrophic events appear to reflect basic DNA metabolism operative throughout an organism's life cycle.

TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

15q26.3 deletions distal to IGF1R cause growth retardation, congenital heart defect and skeletal anomalies: Case report and review of literature.

15q26 deletion is a rare genomic disorder characterized by intrauterine and postnatal growth retardation, microcephaly, intellectual disability, and congenital malformations. Here, we report a 4-month-old female with intrauterine growth retardation, short stature, pulmonary hypertension, atrial septal defect and congenital bowing of long bones of the legs. Chromosomal microarray analysis showed a de novo deletion of approximately 2.1 Mb at 15q26.3 region that does not include IGF1R. Our analysis of patients documented in the literature and the DECIPHER database with 15q26 deletions distal to IGF1R, including 10 patients with de novo pure deletions, allowed us to define the smallest region of overlap to 686 kb. This region includes ALDH1A3, LRRK1, CHSY1, SELENOS, SNRPA1, and PCSK6. We propose haploinsufficiency of one or more genes, besides IGF1R, within this region may contribute to the clinical findings in patients with 15q26.3 deletion.

De Novo Missense Substitutions in the Gene Encoding CDK8, a Regulator of the Mediator Complex, Cause a Syndromic Developmental Disorder.

The Mediator is an evolutionarily conserved, multi-subunit complex that regulates multiple steps of transcription. Mediator activity is regulated by the reversible association of a four-subunit module comprising CDK8 or CDK19 kinases, together with cyclin C, MED12 or MED12L, and MED13 or MED13L. Mutations in MED12, MED13, and MED13L were previously identified in syndromic developmental disorders with overlapping phenotypes. Here, we report CDK8 mutations (located at 13q12.13) that cause a phenotypically related disorder. Using whole-exome or whole-genome sequencing, and by international collaboration, we identified eight different heterozygous missense CDK8 substitutions, including 10 shown to have arisen de novo, in 12 unrelated subjects; a recurrent mutation, c.185C>T (p.Ser62Leu), was present in five individuals. All predicted substitutions localize to the ATP-binding pocket of the kinase domain. Affected individuals have overlapping phenotypes characterized by hypotonia, mild to moderate intellectual disability, behavioral disorders, and variable facial dysmorphism. Congenital heart disease occurred in six subjects; additional features present in multiple individuals included agenesis of the corpus callosum, ano-rectal malformations, seizures, and hearing or visual impairments. To evaluate the functional impact of the mutations, we measured phosphorylation at STAT1-Ser727, a known CDK8 substrate, in a CDK8 and CDK19 CRISPR double-knockout cell line transfected with wild-type (WT) or mutant CDK8 constructs. These experiments demonstrated a reduction in STAT1 phosphorylation by all mutants, in most cases to a similar extent as in a kinase-dead control. We conclude that missense mutations in CDK8 cause a developmental disorder that has phenotypic similarity to syndromes associated with mutations in other subunits of the Mediator kinase module, indicating probable overlap in pathogenic mechanisms.

Expanded phenotype of AARS1-related white matter disease.

PURPOSE: Recent reports of individuals with cytoplasmic transfer RNA (tRNA) synthetase-related disorders have identified cases with phenotypic variability from the index presentations. We sought to assess phenotypic variability in individuals with AARS1-related disease. METHODS: A cross-sectional survey was performed on individuals with biallelic variants in AARS1. Clinical data, neuroimaging, and genetic testing results were reviewed. Alanyl tRNA synthetase (AlaRS) activity was measured in available fibroblasts. RESULTS: We identified 11 affected individuals. Two phenotypic presentations emerged, one with early infantile-onset disease resembling the index cases of AARS1-related epileptic encephalopathy with deficient myelination (n = 7). The second (n = 4) was a later-onset disorder, where disease onset occurred after the first year of life and was characterized on neuroimaging by a progressive posterior predominant leukoencephalopathy evolving to include the frontal white matter. AlaRS activity was significantly reduced in five affected individuals with both early infantile-onset and late-onset phenotypes. CONCLUSION: We suggest that variants in AARS1 result in a broader clinical spectrum than previously appreciated. The predominant form results in early infantile-onset disease with epileptic encephalopathy and deficient myelination. However, a subgroup of affected individuals manifests with late-onset disease and similarly rapid progressive clinical decline. Longitudinal imaging and clinical follow-up will be valuable in understanding factors affecting disease progression and outcome.

A phenotypically diverse family with an atypical 22q11.2 deletion due to an unbalanced 18q23;22q11.2 translocation.

The 22q11.2 deletion syndrome (22q11.2 DS) is the most common deletion syndrome in humans. In most cases, it occurs de novo. A rare family of three with 22q11.2 deletion syndrome (22q11.2 DS) resulting from an unbalanced 18q;22q translocation is reported here. Their deletion region is atypical in that it includes only 26 of the 36 genes in the minimal critical 22q11.2 DS region but it involves the loss of the centromeric 22q region and the entire p arm. The deletion region overlaps with seven other rare atypical cases; common to all cases was the loss of a region including SEPT5-GP1BB proximally and most of ARVCF distally. Interrogation of the deleted 22q region proximal to the canonical 22q11.2 deletion region in the DECIPHER database showed seven cases with isolated or combined traits of 22q11.2 DS, including three with clefts. The phenotypes in the present family thus may result from the loss of a subset of genes in the critical region, or alternatively the loss of other genes or sequences in the proximal 22q deletion region, or interactive effects among these. Despite the identical deletion locus in the three affected family members, expression of the 22q11.2 DS traits differed substantially among them. These three related cases thus contribute to knowledge of 22q11.2 DS in that their unusual deletion locus co-occurred with the cardinal features of the syndrome while their identical deletions are associated with variable phenotypic expression.

An assessment of the role of vinculin loss of function variants in inherited cardiomyopathy.

The ACMG/AMP variant classification framework was intended for highly penetrant Mendelian conditions. While it is appreciated that clinically relevant variants exhibit a wide spectrum of penetrance, accurately assessing and expressing the pathogenicity of variants with lower penetrance can be challenging. The vinculin (VCL) gene illustrates these challenges. Model organism data provide evidence that loss of function of VCL may play a role in cardiomyopathy and aggregate case-control studies suggest low penetrance. VCL loss of function variants, however, are rarely identified in affected probands and therefore there is a paucity of family studies clarifying the clinical significance of individual variants. This study, which aggregated data from >18,000 individuals who underwent gene panel or exome testing for inherited cardiomyopathies, identified 32 probands with VCL loss-of-function variants and confirmed enrichment in probands with dilated cardiomyopathy (odds ratio [OR] = 9.01; confidence interval [CI] = 4.93-16.45). Our data revealed that the majority of these individuals (89.5%) had pediatric onset of disease. Family studies demonstrated that heterozygous loss of function of VCL alone is insufficient to cause cardiomyopathy but that these variants do contribute to disease risk. In conclusion, VCL loss-of-function variants should be reported in a diagnostic setting but need to be clearly distinguished as having lower penetrance.

Overlapping SETBP1 gain-of-function mutations in Schinzel-Giedion syndrome and hematologic malignancies.

Schinzel-Giedion syndrome (SGS) is a rare developmental disorder characterized by multiple malformations, severe neurological alterations and increased risk of malignancy. SGS is caused by de novo germline mutations clustering to a 12bp hotspot in exon 4 of SETBP1. Mutations in this hotspot disrupt a degron, a signal for the regulation of protein degradation, and lead to the accumulation of SETBP1 protein. Overlapping SETBP1 hotspot mutations have been observed recurrently as somatic events in leukemia. We collected clinical information of 47 SGS patients (including 26 novel cases) with germline SETBP1 mutations and of four individuals with a milder phenotype caused by de novo germline mutations adjacent to the SETBP1 hotspot. Different mutations within and around the SETBP1 hotspot have varying effects on SETBP1 stability and protein levels in vitro and in in silico modeling. Substitutions in SETBP1 residue I871 result in a weak increase in protein levels and mutations affecting this residue are significantly more frequent in SGS than in leukemia. On the other hand, substitutions in residue D868 lead to the largest increase in protein levels. Individuals with germline mutations affecting D868 have enhanced cell proliferation in vitro and higher incidence of cancer compared to patients with other germline SETBP1 mutations. Our findings substantiate that, despite their overlap, somatic SETBP1 mutations driving malignancy are more disruptive to the degron than germline SETBP1 mutations causing SGS. Additionally, this suggests that the functional threshold for the development of cancer driven by the disruption of the SETBP1 degron is higher than for the alteration in prenatal development in SGS. Drawing on previous studies of somatic SETBP1 mutations in leukemia, our results reveal a genotype-phenotype correlation in germline SETBP1 mutations spanning a molecular, cellular and clinical phenotype.

FOXG1 syndrome: genotype-phenotype association in 83 patients with FOXG1 variants.

PurposeThe study aimed at widening the clinical and genetic spectrum and assessing genotype-phenotype associations in FOXG1 syndrome due to FOXG1 variants.MethodsWe compiled 30 new and 53 reported patients with a heterozygous pathogenic or likely pathogenic variant in FOXG1. We grouped patients according to type and location of the variant. Statistical analysis of molecular and clinical data was performed using Fisher's exact test and a nonparametric multivariate test.ResultsAmong the 30 new patients, we identified 19 novel FOXG1 variants. Among the total group of 83 patients, there were 54 variants: 20 frameshift (37%), 17 missense (31%), 15 nonsense (28%), and 2 in-frame variants (4%). Frameshift and nonsense variants are distributed over all FOXG1 protein domains; missense variants cluster within the conserved forkhead domain. We found a higher phenotypic variability than previously described. Genotype-phenotype association revealed significant differences in psychomotor development and neurological features between FOXG1 genotype groups. More severe phenotypes were associated with truncating FOXG1 variants in the N-terminal domain and the forkhead domain (except conserved site 1) and milder phenotypes with missense variants in the forkhead conserved site 1.ConclusionsThese data may serve for improved interpretation of new FOXG1 sequence variants and well-founded genetic counseling.

WRN Mutation Update: Mutation Spectrum, Patient Registries, and Translational Prospects.

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by a constellation of adult onset phenotypes consistent with an acceleration of intrinsic biological aging. It is caused by pathogenic variants in the WRN gene, which encodes a multifunctional nuclear protein with exonuclease and helicase activities. WRN protein is thought to be involved in optimization of various aspects of DNA metabolism, including DNA repair, recombination, replication, and transcription. In this update, we summarize a total of 83 different WRN mutations, including eight previously unpublished mutations identified by the International Registry of Werner Syndrome (Seattle, WA) and the Japanese Werner Consortium (Chiba, Japan), as well as 75 mutations already reported in the literature. The Seattle International Registry recruits patients from all over the world to investigate genetic causes of a wide variety of progeroid syndromes in order to contribute to the knowledge of basic mechanisms of human aging. Given the unusually high prevalence of WS patients and heterozygous carriers in Japan, the major goal of the Japanese Consortium is to develop effective therapies and to establish management guidelines for WS patients in Japan and elsewhere. This review will also discuss potential translational approaches to this disorder, including those currently under investigation.

Chronic pain in Noonan Syndrome: A previously unreported but common symptom.

Noonan syndrome (NS) is a multiple malformation syndrome characterized by pulmonic stenosis, cardiomyopathy, short stature, lymphatic dysplasia, craniofacial anomalies, cryptorchidism, clotting disorders, and learning disabilities. Eight genes in the RAS/MAPK signaling pathway are implicated in NS. Chronic pain is an uncommon feature. To investigate the prevalence of pain in NS, we distributed a two-part questionnaire about pain among NS individuals at the Third International Meeting on Genetic Syndromes of the Ras/MAPK Pathway. The first part of the questionnaire queried demographic information among all NS participants. The second part was completed by individuals with chronic pain. Questions included musculoskeletal problems and clinical features of pain. Forty-five questionnaires were analyzed; 53% of subjects were female. Mean age was 17 (2-48) years; 47% had a PTPN11 mutation. Sixty-two percent (28/45) of individuals with NS experienced chronic pain. There was a significant relationship between prevalence of pain and residing in a cold climate (P = 0.004). Pain occurred commonly in extremities/joints and head/trunk, but more commonly in extremities/joints (P = 0.066). Subjects with hypermobile joints were more likely to have pain (P = 0.052). Human growth hormone treatment was not statistically significant among subjects without chronic pain (P = 0.607). We conclude that pain is a frequent and under-recognized clinical feature of NS. Chronic pain may be associated with joint hypermobility and aggravated by colder climate. Our study is a preliminary investigation that should raise awareness about pain as a common symptom in children and adults with NS.

A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder.

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway - KYNU, HAAO, and NADSYN1 - have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain-specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/- mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1-/- mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Structural brain anomalies in patients with FOXG1 syndrome and in Foxg1+/- mice.

OBJECTIVE: FOXG1 syndrome is a rare neurodevelopmental disorder associated with heterozygous FOXG1 variants or chromosomal microaberrations in 14q12. The study aimed at assessing the scope of structural cerebral anomalies revealed by neuroimaging to delineate the genotype and neuroimaging phenotype associations. METHODS: We compiled 34 patients with a heterozygous (likely) pathogenic FOXG1 variant. Qualitative assessment of cerebral anomalies was performed by standardized re-analysis of all 34 MRI data sets. Statistical analysis of genetic, clinical and neuroimaging data were performed. We quantified clinical and neuroimaging phenotypes using severity scores. Telencephalic phenotypes of adult Foxg1+/- mice were examined using immunohistological stainings followed by quantitative evaluation of structural anomalies. RESULTS: Characteristic neuroimaging features included corpus callosum anomalies (82%), thickening of the fornix (74%), simplified gyral pattern (56%), enlargement of inner CSF spaces (44%), hypoplasia of basal ganglia (38%), and hypoplasia of frontal lobes (29%). We observed a marked, filiform thinning of the rostrum as recurrent highly typical pattern of corpus callosum anomaly in combination with distinct thickening of the fornix as a characteristic feature. Thickening of the fornices was not reported previously in FOXG1 syndrome. Simplified gyral pattern occurred significantly more frequently in patients with early truncating variants. Higher clinical severity scores were significantly associated with higher neuroimaging severity scores. Modeling of Foxg1 heterozygosity in mouse brain recapitulated the associated abnormal cerebral morphology phenotypes, including the striking enlargement of the fornix. INTERPRETATION: Combination of specific corpus callosum anomalies with simplified gyral pattern and hyperplasia of the fornices is highly characteristic for FOXG1 syndrome.

The mitochondrial 13513G>A mutation is associated with Leigh disease phenotypes independent of complex I deficiency in muscle.

The mitochondrial 13513G>A (D393N) mutation in the ND5 subunit of the respiratory chain complex I was initially described in association with MELAS syndrome. Recent observations have linked this mutation to Leigh disease. We screened for the 13513G>A mutation in a cohort of 265 patients with Leigh and Leigh-like disease. The mutation was found in a total of 5 patients. An additional patient who had clinical presentation consistent with a Leigh-like phenotype but with a normal brain MRI was added to the cohort. None of an additional 88 patients meeting MELAS disease criteria, nor 56 patients with respiratory chain deficiency screened for the 13513G>A were found positive for the mutation. The most frequent clinical manifestations in our patients were hypotonia, ocular and cerebellar involvement. Low mutation heteroplasmy in the range of 20-40% was observed in all 6 patients. This observation is consistent with the previously reported low heteroplasmy of this mutation in some patients with the 13513G>A mutation and complex I deficiency. However, normal complex I activity was observed in two patients in our cohort. As most patients with Leigh-like disease and the 13513G>A mutation have been described with complex I deficiency, this report adds to the previously reported subset of patients with normal respiratory complex function. We conclude that in any patient with Leigh or Leigh-like disease, testing for the 13513G>A mutation is clinically relevant and low mutant loads in blood or muscle may be considered pathogenic, in the presence of normal respiratory chain enzyme activities.

Progressive Movement Disorder in Brothers Carrying a GNAO1 Mutation Responsive to Deep Brain Stimulation.

GNAO1, located on chromosome 16q12.2, encodes for 1 of the heterotrimeric guanine binding proteins subunits (G proteins), specifically Gαo, which has been implicated as having an important role in brain function. GNAO1 mutations have been shown to impart oncogene properties as well as cause epileptic encephalopathy. The authors report 2 cases of brothers with a severe movement disorder and hypotonia without epilepsy who have been confirmed by whole exome sequencing to have a novel mutation in GNAO1. Their movement disorder improved significantly with deep brain stimulation.