Biallelic variants in GTF3C5, a regulator of RNA polymerase III-mediated transcription, cause a multisystem developmental disorder.

General transcription factor IIIC subunit 5 (GTF3C5) encodes transcription factor IIIC63 (TFIIIC63). It binds to DNA to recruit another transcription factor, TFIIIB, and RNA polymerase III (Pol III) to mediate the transcription of small noncoding RNAs, such as tRNAs. Here, we report four individuals from three families presenting with a multisystem developmental disorder phenotype with biallelic variants in GTF3C5. The overlapping features include growth retardation, developmental delay, intellectual disability, dental anomalies, cerebellar malformations, delayed bone age, skeletal anomalies, and facial dysmorphism. Using lymphoblastoid cell lines (LCLs) from two affected individuals, we observed a reduction in TFIIIC63 protein levels compared to control LCLs. Genome binding of TFIIIC63 protein is also reduced in LCL from one of the affected individuals. Additionally, approximately 40% of Pol III binding regions exhibited reduction in the level of Pol III occupancy in the mutant genome relative to the control, while approximately 54% of target regions showed comparable levels of Pol III occupancy between the two, indicating partial impairment of Pol III occupancy in the mutant genome. Yeasts with subject-specific variants showed temperature sensitivity and impaired growth, supporting the notion that the identified variants have deleterious effects. gtf3c5 mutant zebrafish showed developmental defects, including a smaller body, head, and eyes. Taken together, our data show that GTF3C5 plays an important role in embryonic development, and that biallelic variants in this gene cause a multisystem developmental disorder. Our study adds GTF3C5-related disorder to the growing list of genetic disorders associated with Pol III transcription machinery.

Inherited bone marrow failure with macrothrombocytopenia due to germline tubulin beta class I (TUBB) variant.

Germline mutations in tubulin beta class I (TUBB), which encodes one of the ß-tubulin isoforms, were previously associated with neurological and cutaneous abnormalities. Here, we describe the first case of inherited bone marrow (BM) failure, including marked thrombocytopenia, morphological abnormalities, and cortical dysplasia, associated with a de novo p.D249V variant in TUBB. Mutant TUBB had abnormal cellular localisation in transfected cells. Following interferon/ribavirin therapy administered for transfusion-acquired hepatitis C, severe pancytopenia and BM aplasia ensued, which was unresponsive to immunosuppression. Acquired chromosome arm 6p loss of heterozygosity was identified, leading to somatic loss of the mutant TUBB allele.

How parents of children with ataxia-telangiectasia use dynamic coping to navigate cyclical uncertainty.

Ataxia-telangiectasia (A-T) is a rare, childhood-onset, multi-systemic, progressive condition. Parents of children with rare diseases like A-T are emotionally, socially, and psychologically impacted by the diagnosis. To examine the parental perspective of having a child with A-T, and to better understand how parents cope with an A-T diagnosis, we conducted 10 semistructured interviews. Thematic analysis using a phenomenological approach resulted in five themes: (1) Parental responsibilities change as the result of an A-T diagnosis, (2) An A-T diagnosis brings about shifts in identity for all family members, (3) Parental coping changes over time, (4) A-T parents experience continuous uncertainty and a lack of stability, and (5) A-T parents receive support from various people, places, and resources. Many parents fostered resilience by adopting a present-centered and positive mindset about the impacts of the diagnosis. Parents also became A-T experts and used their knowledge to advocate for their children and help mentor other parents. Responses from parents indicated a need for providers to incorporate parental mental well-being check-ins to pediatric rare disease appointments and welcome parents as respected members of their children's care team. Genetic counselors are in a unique position to help coordinate complex care for children with A-T (and other rare diseases) and provide support to family members using the framework of family-centered care. This paper offers suggestions for expanding support and learning to cope with a difficult diagnosis for parents of children with rare diseases, specifically A-T, based on stories from parents of children with A-T.

Diagnostic testing laboratories are valuable partners for disease gene discovery: 5-year experience with GeneMatcher.

Although the rates of disease gene discovery have steadily increased with the expanding use of genome and exome sequencing by clinical and research laboratories, only ~16% of genes in the genome have confirmed disease associations. Here we describe our clinical laboratory's experience utilizing GeneMatcher, an online portal designed to promote disease gene discovery and data sharing. Since 2016, we submitted 246 candidates from 243 unique genes to GeneMatcher, of which 111 (45%) are now clinically characterized. Submissions meeting our candidate gene-reporting criteria based on a scoring system using patient and molecular-weighted evidence were significantly more likely to be characterized as of October 2021 versus genes that did not meet our clinical-reporting criteria (p = 0.025). We reported relevant findings related to these newly characterized gene-disease associations in 477 probands. In 218 (46%) instances, we issued reclassifications after an initial negative or candidate gene (uncertain) report. We coauthored 104 publications delineating gene-disease relationships, including descriptions of new associations (60%), additional supportive evidence (13%), subsequent descriptive cohorts (23%), and phenotypic expansions (4%). Clinical laboratories are pivotal for disease gene discovery efforts and can screen phenotypes based on genotype matches, contact clinicians of relevant cases, and issue proactive reclassification reports.

Misattributed parentage identified through diagnostic exome sequencing: Frequency of detection and reporting practices.

Access to genetic testing, namely, diagnostic exome sequencing (DES), has significantly improved, subsequently increasing the likelihood of discovering incidental findings, such as misattributed relationships and specifically misattributed parentage (MP). Until the recently published ACMG statement, there had been no consensus for laboratories and clinicians to follow when addressing such findings. Family-based genomic testing is valuable for accurate variant interpretation but has the potential to uncover misattributed familial relationships. Here, we present the first published data on the frequency of MP identified through DES at a clinical laboratory. We also investigated clinicians' decisions on how to proceed with analysis, reporting, and disclosure. A database of 6,752 families who underwent parent-proband ('trio') DES was retrospectively reviewed for molecular identification of MP and clinicians' MP disclosure decisions. Among 6,752 trios, 39 cases of MP were detected (0.58%). Non-paternity was detected in all cases, and in one instance, non-maternity was also identified. All clinicians decided to proceed by omitting the MP individual from the analysis. Clinicians chose to proceed with duo analysis (87.2%), modify information on the report (74.4%), and communicate MP results to the mother (71.8%), suggesting a trend toward not disclosing to the putative father or proband. The data show that trio DES involves a chance of detecting MP and that clinician disclosure practices do not appear to routinely include direct disclosure to the putative father. MP identified in our parent-proband trios sent in for DES is lower than the reported frequency of MP in the general population due in part to ascertainment bias as families with known or suspected MP are presumably less likely to pursue trio testing. These data may inform laboratory policies and clinician practices for addressing incidental findings such as MP.

Backpack health reduces data-sharing barriers between the medical community and individuals with rare diseases.

Technology has changed the way we approach medical care: health data is constantly being generated, medical discoveries are progressing more rapidly, and individuals are more connected across the world than ever before. Backpack Health is a global personal health record platform that harnesses the power of technology to connect users to their primary health data sources, the medical community, and researchers. By syncing with existing patient portals, health data can be stored on the Backpack Health platform and easily accessed and controlled by users in one connected interface. Individuals manage and collate their current and past conditions, genetic test results, symptoms, medications, procedures, labs, and other health data. Users are empowered to disseminate their information to clinicians, researchers, foundations, and pharmaceutical and biotechnology companies they connect with through the Backpack Health application. Here, we describe how two rare disease advocacy groups, The Marfan Foundation and Project Alive, utilize Backpack Health to connect with their target populations. Through secure transfer of pseudonymized data, groups can query their members to improve understanding of clinical features and to facilitate meaningful research. Responses to the groups' surveys show strong member engagement with high completion rates and increases in new Backpack Health users when surveys are deployed. Data from these surveys have been published and used to better inform clinical outcomes for treatment trials. By connecting users directly to the foundations, clinicians, researchers, and industry partners working on their condition, Backpack Health is instrumental in fast-tracking medical discoveries and treatment for rare diseases.

De Novo Mutations in Protein Kinase Genes CAMK2A and CAMK2B Cause Intellectual Disability.

Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway.

Clinical diagnostic exome sequencing in dystonia: Genetic testing challenges for complex conditions.

Patients with dystonia are particularly appropriate for diagnostic exome sequencing (DES), due to the complex, diverse features and genetic heterogeneity. Personal and family history data were collected from test requisition forms and medical records from 189 patients with reported dystonia and available family members received for clinical DES. Of them, 20.2% patients had a positive genetic finding associated with dystonia. Detection rates for cases with isolated and combined dystonia were 22.4% and 25.0%, respectively. 71.4% of the cohort had co-occurring non-movement-related findings and a detection rate of 24.4%. Patients with childhood-onset dystonia trended toward higher detection rates (31.8%) compared to infancy (23.6%), adolescence (12.5%), and early-adulthood onset (16%). Uncharacterized gene findings were found in 6.7% (8/119) of cases that underwent analysis for genes without an established disease relationship. Patients with intellectual disability/developmental delay, seizures/epilepsy and/or multifocal dystonia were more likely to have positive findings (P = .0093, .0397, .0006). Four (2.1%) patients had findings in two genes, and seven (3.7%) had reclassification after the original report due to new literature, new clinical information or reanalysis request. Pediatric patients were more likely to have positive findings (P = .0180). Our observations show utility of family-based DES in patients with dystonia and illustrate the complexity of testing.

Genetic disorders and mortality in infancy and early childhood: delayed diagnoses and missed opportunities.

PURPOSE: Infants admitted to a level IV neonatal intensive care unit (NICU) who do not survive early childhood are a population that is probably enriched for rare genetic disease; we therefore characterized their genetic diagnostic evaluation. METHODS: This is a retrospective analysis of infants admitted to our NICU between 1 January 2011 and 31 December 2015 who were deceased at the time of records review, with age at death less than 5 years. RESULTS: A total of 2,670 infants were admitted; 170 later died. One hundred six of 170 (62%) had an evaluation for a genetic or metabolic disorder. Forty-seven of 170 (28%) had laboratory-confirmed genetic diagnoses, although 14/47 (30%) diagnoses were made postmortem. Infants evaluated for a genetic disorder spent more time in the NICU (median 13.5 vs. 5.0 days; p = 0.003), were older at death (median 92.0 vs. 17.5 days; p < 0.001), and had similarly high rates of redirection of care (86% vs. 79%; p = 0.28). CONCLUSION: Genetic disorders were suspected in many infants but found in a minority. Approximately one-third of diagnosed infants died before a laboratory-confirmed genetic diagnosis was made. This highlights the need to improve genetic diagnostic evaluation in the NICU, particularly to support end-of-life decision making.

Three-generation family with novel contiguous gene deletion on chromosome 2p22 associated with thoracic aortic aneurysm syndrome.

Latent transforming growth factor binding proteins (LTBP) are a family of extracellular matrix glycoproteins that play an important role in the regulation of transforming growth factor beta (TGF-ß) activation. Dysregulation of the TGF-ß pathway has been implicated in the pathogenesis of inherited disorders predisposing to thoracic aortic aneurysms syndromes (TAAS) including Marfan syndrome (MFS; FBN1) and Loeys-Dietz syndrome (LDS; TGFBR1, TGFBR2, TGFB2, TGFB3, SMAD2, SMAD3). While these syndromes have distinct clinical criteria, they share clinical features including aortic root dilation and musculoskeletal findings. LTBP1 is a component of the TGF-ß pathway that binds to fibrillin-1 in the extracellular matrix rendering TGF-ß inactive. We describe a three-generation family case series with a heterozygous ∼5.1 Mb novel contiguous gene deletion of chromosome 2p22.3-p22.2 involving 11 genes, including LTBP1. The deletion has been identified in the proband, father and grandfather, who all have a phenotype consistent with a TAAS. Findings include thoracic aortic dilation, ptosis, malar hypoplasia, high arched palate, retrognathia, pes planus, hindfoot deformity, obstructive sleep apnea, and low truncal tone during childhood with joint laxity that progressed to reduced joint mobility over time. While the three affected individuals did not meet criteria for either MFS or LDS, they shared features of both. Although the deletion includes 11 genes, given the relationship between LTBP1, TGF-ß, and fibrillin-1, LTBP1 stands out as one of the possible candidate genes for the clinical syndrome observed in this family. More studies are necessary to evaluate the potential role of LTBP1 in the pathophysiology of TAAS.

The Epithelial Sodium Channel Is a Modifier of the Long-Term Nonprogressive Phenotype Associated with F508del CFTR Mutations.

Cystic fibrosis (CF) remains the most lethal genetic disease in the Caucasian population. However, there is great variability in clinical phenotypes and survival times, even among patients harboring the same genotype. We identified five patients with CF and a homozygous F508del mutation in the CFTR gene who were in their fifth or sixth decade of life and had shown minimal changes in lung function over a longitudinal period of more than 20 years. Because of the rarity of this long-term nonprogressive phenotype, we hypothesized these individuals may carry rare genetic variants in modifier genes that ameliorate disease severity. Individuals at the extremes of survival time and lung-function trajectory underwent whole-exome sequencing, and the sequencing data were filtered to include rare missense, stopgain, indel, and splicing variants present with a mean allele frequency of <0.2% in general population databases. Epithelial sodium channel (ENaC) mutants were generated via site-directed mutagenesis and expressed for Xenopus oocyte assays. Four of the five individuals carried extremely rare or never reported variants in the SCNN1D and SCNN1B genes of the ENaC. Separately, an independently enriched rare variant in SCNN1D was identified in the Exome Variant Server database associated with a milder pulmonary disease phenotype. Functional analysis using Xenopus oocytes revealed that two of the three variants in δ-ENaC encoded by SCNN1D exhibited hypomorphic channel activity. Our data suggest a potential role for δ-ENaC in controlling sodium reabsorption in the airways, and advance the plausibility of ENaC as a therapeutic target in CF.

Congenital sideroblastic anemia due to mutations in the mitochondrial HSP70 homologue HSPA9.

The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases characterized by defects in mitochondrial heme synthesis, iron-sulfur (Fe-S) cluster biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homolog located in the chromosome 5q deletion syndrome 5q33 critical deletion interval and involved in mitochondrial Fe-S biogenesis, result in CSA inherited as an autosomal recessive trait. In a fraction of patients with just 1 severe loss-of-function allele, expression of the clinical phenotype is associated with a common coding single nucleotide polymorphism in trans that correlates with reduced messenger RNA expression and results in a pseudodominant pattern of inheritance.

Novel mutation in CNTNAP1 results in congenital hypomyelinating neuropathy.

INTRODUCTION: Congenital hypomyelinating neuropathy (CHN) is a rare congenital neuropathy that presents in the neonatal period and has been linked previously to mutations in several genes associated with myelination. A recent study has linked 4 homozygous frameshift mutations in the contactin-associated protein 1 (CNTNAP1) gene with this condition. METHODS: We report a neonate with CHN who was found to have absent sensory nerve and compound muscle action potentials and hypomyelination on nerve biopsy. RESULTS: On whole exome sequencing, we identified a novel CNTNAP1 homozygous missense mutation (p.Arg388Pro) in the proband, and both parents were carriers. Molecular modeling suggests that this variant disrupts a ß-strand to cause an unstable structure and likely significant changes in protein function. CONCLUSIONS: This report links a missense CNTNAP1 variant to the disease phenotype previously associated only with frameshift mutations. Muscle Nerve 55: 761-765, 2017.

Clinical heterogeneity associated with KCNA1 mutations include cataplexy and nonataxic presentations.

Mutations in the KCNA1 gene are known to cause episodic ataxia/myokymia syndrome type 1 (EA1). Here, we describe two families with unique presentations who were enrolled in an IRB-approved study, extensively phenotyped, and whole exome sequencing (WES) performed. Family 1 had a diagnosis of isolated cataplexy triggered by sudden physical exertion in multiple affected individuals with heterogeneous neurological findings. All enrolled affected members carried a KCNA1 c.941T>C (p.I314T) mutation. Family 2 had an 8-year-old patient with muscle spasms with rigidity for whom WES revealed a previously reported heterozygous missense mutation in KCNA1 c.677C>G (p.T226R), confirming the diagnosis of EA1 without ataxia. WES identified variants in KCNA1 that explain both phenotypes expanding the phenotypic spectrum of diseases associated with mutations of this gene. KCNA1 mutations should be considered in patients of all ages with episodic neurological phenotypes, even when ataxia is not present. This is an example of the power of genomic approaches to identify pathogenic mutations in unsuspected genes responsible for heterogeneous diseases.

Overlapping 16p13.11 deletion and gain of copies variations associated with childhood onset psychosis include genes with mechanistic implications for autism associated pathways: Two case reports.

Copy number variability at 16p13.11 has been associated with intellectual disability, autism, schizophrenia, epilepsy, and attention-deficit hyperactivity disorder. Adolescent/adult- onset psychosis has been reported in a subset of these cases. Here, we report on two children with CNVs in 16p13.11 that developed psychosis before the age of 7. The genotype and neuropsychiatric abnormalities of these patients highlight several overlapping genes that have possible mechanistic relevance to pathways previously implicated in Autism Spectrum Disorders, including the mTOR signaling and the ubiquitin-proteasome cascades. A careful screening of the 16p13.11 region is warranted in patients with childhood onset psychosis.

Novel variants in KAT6B spectrum of disorders expand our knowledge of clinical manifestations and molecular mechanisms.

The phenotypic variability associated with pathogenic variants in Lysine Acetyltransferase 6B (KAT6B, a.k.a. MORF, MYST4) results in several interrelated syndromes including Say-Barber-Biesecker-Young-Simpson Syndrome and Genitopatellar Syndrome. Here we present 20 new cases representing 10 novel KAT6B variants. These patients exhibit a range of clinical phenotypes including intellectual disability, mobility and language difficulties, craniofacial dysmorphology, and skeletal anomalies. Given the range of features previously described for KAT6B-related syndromes, we have identified additional phenotypes including concern for keratoconus, sensitivity to light or noise, recurring infections, and fractures in greater numbers than previously reported. We surveyed clinicians to qualitatively assess the ways families engage with genetic counselors upon diagnosis. We found that 56% (10/18) of individuals receive diagnoses before the age of 2 years (median age = 1.96 years), making it challenging to address future complications with limited accessible information and vast phenotypic severity. We used CRISPR to introduce truncating variants into the KAT6B gene in model cell lines and performed chromatin accessibility and transcriptome sequencing to identify key dysregulated pathways. This study expands the clinical spectrum and addresses the challenges to management and genetic counseling for patients with KAT6B-related disorders.

Expanding the phenotypic spectrum associated with OPHN1 variants.

Genomic sequencing has allowed for the characterization of new gene-to-disease relationships, as well as the identification of variants in established disease genes in patients who do not fit the classically-described phenotype. This is especially true in rare syndromes where the clinical spectrum is not fully known. After a lengthy and costly diagnostic odyssey, patients with atypical presentations may be left with many questions even after a genetic diagnosis is identified. We present a 22-year old male with hypotonia, developmental delay, seizure disorder, and dysmorphic facial features who enrolled in our rare disease research center at 18 years of age, where exome sequencing revealed a novel, likely pathogenic variant in the OPHN1 gene. Through efforts by the study team and collaborations with the larger genetics community, contacts with other families with OPHN1 variants were eventually made, and outreach by these families expanded the patient network. This partnership between families and researchers facilitated the gathering of phenotypic information, allowing for comparison of clinical presentations among three new patients and those previously reported in the literature. These comparisons found previously unreported commonalities between the newly identified patients, such as the presence of otitis media and the lack of genitourinary abnormalities (i.e. hypoplastic scrotum, microphallus, cryptorchidism), which had been noted to be classic features of patients with OPHN1 variants. As genomic sequencing becomes more common, connecting patients with novel variants in the same gene will facilitate phenotypic analysis and continue to refine the clinical spectrum associated with that gene.

Beta-Ketothiolase Deficiency Presenting with Metabolic Stroke After a Normal Newborn Screen in Two Individuals.

Beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase) deficiency is a genetic disorder characterized by impaired isoleucine catabolism and ketone body utilization that predisposes to episodic ketoacidosis. It results from biallelic pathogenic variants in the ACAT1 gene, encoding mitochondrial beta-ketothiolase. We report two cases of beta-ketothiolase deficiency presenting with acute ketoacidosis and "metabolic stroke." The first patient presented at 28 months of age with metabolic acidosis and pallidal stroke in the setting of a febrile gastrointestinal illness. Although 2-methyl-3-hydroxybutyric acid and trace quantities of tiglylglycine were present in urine, a diagnosis of glutaric acidemia type I was initially suspected due to the presence of glutaric and 3-hydroxyglutaric acids. A diagnosis of beta-ketothiolase deficiency was ultimately made through whole exome sequencing which revealed compound heterozygous variants in ACAT1. Fibroblast studies for beta-ketothiolase enzyme activity were confirmatory. The second patient presented at 6 months of age with ketoacidosis, and was found to have elevations of urinary 2-methyl-3-hydroxybutyric acid, 2-methylacetoacetic acid, and tiglylglycine. Sequencing of ACAT1 demonstrated compound heterozygous presumed causative variants. The patient exhibited choreoathethosis 2 months after the acute metabolic decompensation. These cases highlight that, similar to a number of other organic acidemias and mitochondrial disorders, beta-ketothiolase deficiency can present with metabolic stroke. They also illustrate the variability in clinical presentation, imaging, and biochemical evaluation that make screening for and diagnosis of this rare disorder challenging, and further demonstrate the value of whole exome sequencing in the diagnosis of metabolic disorders.

Long-Gap Esophageal Atresia Is a Unique Entity within the Esophageal Atresia Defect Spectrum.

BACKGROUND: Long-gap esophageal atresia (LGEA) may have clinical and syndromic presentations different from those of esophageal atresia (EA) that affects shorter segments of the esophagus (non-LGEA). This may suggest unique underlying developmental mechanisms. OBJECTIVES: We sought to characterize clinical differences between LGEA and non-LGEA by carefully phenotyping a cohort of EA patients, and furthermore to assess molecular genetic findings in a subset of them. METHODS: This is a retrospective cohort study to systematically evaluate clinical and genetic findings in EA infants who presented at our institution over a period of 10 years (2005-2015). RESULTS: Two hundred twenty-nine EA patients were identified, 69 (30%) of whom had LGEA. Tracheoesophageal fistula was present in most non-LGEA patients (158 of 160) but in only 30% of LGEA patients. The VACTERL association was more commonly seen with non-LGEA compared to LGEA (70 vs. 25%; p < 0.001). Further, trisomy 21 was more common in LGEA than in non-LGEA. 25% of LGEA patients had an isolated EA diagnosis without other anomalies, compared to <1% for non-LGEA. Chromosomal microarray analysis showed copy number variations (CNV) in 4 of 39 non-LGEA patients and 0 of 3 LGEA patients. A review of the ClinGen database showed that none of those CNV have been previously described with EA. CONCLUSIONS: LGEA represents a unique type of EA. Compared to non-LGEA, it is more likely to be an isolated defect and associated with trisomy 21. Further, it is less commonly seen with VACTERL anomalies. Our findings suggest the involvement of unique pathways that may be distinct from those causing non-LGEA.