Heterozygous Truncating Variants Research Articles

Toward clinical exomes in diagnostics and management of male infertility

Infertility, affecting ∼10% of men, is predominantly caused by primary spermatogenic failure (SPGF). We screened likely pathogenic and pathogenic (LP/P) variants in 638 candidate genes for male infertility in 521 individuals presenting idiopathic SPGF and 323 normozoospermic men in the ESTAND cohort. Molecular diagnosis was reached for 64 men with SPGF (12%), with findings in 39 genes (6%). The yield did not differ significantly between the subgroups with azoospermia (20/185, 11%), oligozoospermia (18/181, 10%), and primary cryptorchidism with SPGF (26/155, 17%). Notably, 19 of 64 LP/P variants (30%) identified in 28 subjects represented recurrent findings in this study and/or with other male infertility cohorts. NR5A1 was the most frequently affected gene, with seven LP/P variants in six SPGF-affected men and two normozoospermic men. The link to SPGF was validated for recently proposed candidate genes ACTRT1, ASZ1, GLUD2, GREB1L, LEO1, RBM5, ROS1, and TGIF2LY. Heterozygous truncating variants in BNC1, reported in female infertility, emerged as plausible causes of severe oligozoospermia. Data suggested that several infertile men may present congenital conditions with less pronounced or pleiotropic phenotypes affecting the development and function of the reproductive system. Genes regulating the hypothalamic-pituitary-gonadal axis were affected in >30% of subjects with LP/P variants. Six individuals had more than one LP/P variant, including five with two findings from the gene panel. A 4-fold increased prevalence of cancer was observed in men with genetic infertility compared to the general male population (8% vs. 2%; p= 4.4×10-3). Expanding genetic testing in andrology will contribute to the multidisciplinary management of SPGF.

Heterozygous truncating variant of TAOK1 in a boy with periventricular nodular heterotopia: a case report and literature review of TAOK1-related neurodevelopmental disorders

BackgroundThousand and one amino-acid kinase 1 (TAOK1) encodes the MAP3K protein kinase TAO1, which has recently been displayed to be essential for neuronal maturation and cortical differentiation during early brain development. Heterozygous variants in TAOK1 have been reported in children with neurodevelopmental disorders, with or without macrocephaly, hypotonia and mild dysmorphic traits. Literature reports lack evidence of neuronal migration disorders in TAOK1 patients, although studies in animal models suggest this possibility.Case presentationWe provide a clinical description of a child with a neurodevelopmental disorder due to a novel TAOK1 truncating variant, whose brain magnetic resonance imaging displays periventricular nodular heterotopia.ConclusionsTo our knowledge, this is the first report of a neuronal migration disorder in a patient with a TAOK1-related neurodevelopmental disorder, thus supporting the hypothesized pathogenic mechanisms of TAOK1 defects.

Recurrent missense variant identified in two unrelated families with MPZL2-related hearing loss, expanding the variant spectrum associated with DFNB111.

MPZL2-related hearing loss is a rare form of autosomal recessive hearing loss characterized by progressive, mild sloping to severe sensorineural hearing loss. Thirty-five previously reported patients had biallelic truncating variants in MPZL2, with the exception of one patient with a missense variant of uncertain significance and a truncating variant. Here, we describe the clinical characteristics and genotypes of five patients from four families with confirmed MPZL2-related hearing loss. A rare missense likely pathogenic variant [NM_005797.4(MPZL2):c.280C>T,p.(Arg94Trp)] located in exon 3 was confirmed to be in trans with a recurrent pathogenic truncating variant that segregated with hearing loss in three of the patients from two unrelated families. This is the first recurrent likely pathogenic missense variant identified in MPZL2. Apparently milder or later-onset hearing loss associated with rare missense variants in MPZL2 indicates that some missense variants in this gene may cause a milder phenotype than that resulting from homozygous or compound heterozygous truncating variants. This study, along with the identification of truncating loss of function and missense MPZL2 variants in several diverse populations, suggests that MPZL2-related hearing loss may be more common than previously appreciated and demonstrates the need for MPZL2 inclusion in hearing loss testing panels.

DAG1 haploinsufficiency is associated with sporadic and familial isolated or pauci-symptomatic hyperCKemia.

DAG1 encodes for dystroglycan, a key component of the dystrophin-glycoprotein complex (DGC) with a pivotal role in skeletal muscle function and maintenance. Biallelic loss-of-function DAG1 variants cause severe muscular dystrophy and muscle-eye-brain disease. A possible contribution of DAG1 deficiency to milder muscular phenotypes has been suggested. We investigated the genetic background of twelve subjects with persistent mild-to-severe hyperCKemia to dissect the role of DAG1 in this condition. Genetic testing was performed through exome sequencing (ES) or custom NGS panels including various genes involved in a spectrum of muscular disorders. Histopathological and Western blot analyses were performed on muscle biopsy samples obtained from three patients. We identified seven novel heterozygous truncating variants in DAG1 segregating with isolated or pauci-symptomatic hyperCKemia in all families. The variants were rare and predicted to lead to nonsense-mediated mRNA decay or the formation of a truncated transcript. In four cases, DAG1 variants were inherited from similarly affected parents. Histopathological analysis revealed a decreased expression of dystroglycan subunits and Western blot confirmed a significantly reduced expression of beta-dystroglycan in muscle samples. This study supports the pathogenic role of DAG1 haploinsufficiency in isolated or pauci-symptomatic hyperCKemia, with implications for clinical management and genetic counseling.

Cingulin regulates hair cell cuticular plate morphology and is required for hearing in human and mouse.

Cingulin (CGN) is a cytoskeleton-associated protein localized at the apical junctions of epithelial cells. CGN interacts with major cytoskeletal filaments and regulates RhoA activity. However, physiological roles of CGN in development and human diseases are currently unknown. Here, we report a multi-generation family presenting with autosomal dominant non-syndromic hearing loss (ADNSHL) that co-segregates with a CGN heterozygous truncating variant, c.3330delG (p.Leu1110Leufs*17). CGN is normally expressed at the apical cell junctions of the organ of Corti, with enriched localization at hair cell cuticular plates and circumferential belts. In mice, the putative disease-causing mutation results in reduced expression and abnormal subcellular localization of the CGN protein, abolishes its actin polymerization activity, and impairs the normal morphology of hair cell cuticular plates and hair bundles. Hair cell-specific Cgn knockout leads to high-frequency hearing loss. Importantly, Cgn mutation knockin mice display noise-sensitive, progressive hearing loss and outer hair cell degeneration. In summary, we identify CGN c.3330delG as a pathogenic variant for ADNSHL and reveal essential roles of CGN in the maintenance of cochlear hair cell structures and auditory function.

A novel heterozygous truncating variant in the AGO1 gene in an Iranian family with schizophrenia as an unreported symptom.

Intellectual disability (ID) and autism spectrum disorders (ASDs) are the most common developmental disorders in humans. Combined, they affect between 3% and 5% of the population. Although high-throughput genomic methods are rapidly increasing the pool of ASD genes, many cases remain idiopathic. AGO1 is one of the less-known genes related to ID/ASD. This gene encodes a core member protein of the RNA-induced silencing complex, which suppresses mRNA expression through cleavage, degradation, and/or translational repression. Generally, patients with defects in the AGO1 gene manifest varying degrees of ID, speech delay, and autistic behaviors. Herein, we used whole-exome sequencing (WES) to investigate an Iranian family with two affected members one of whom manifested ID and autism and the other showed borderline ID and schizophrenia. WES analysis identified a novel heterozygous truncating variant (NM_012199.5:c.1298G>A, p.Trp433Ter) in the AGO1 gene that co-segregated with the phenotypes using Sanger sequencing. Moreover, the structural analysis showed that due to this variant, two critical domains (Mid and PIWI) of the AGO1 protein have been lost, which has a detrimental effect on the protein's function and structure. To the best of our knowledge, schizophrenia has not been reported in patients with AGO1 deficiency, which is a novel phenotypic finding that expands the AGO1-related behavioral disorders. Moreover, this study's findings determined that patients with the same variant in the AGO1 gene may show heterogeneity in manifested phenotypes.

Ablation of the carboxy-terminal end of MAMDC2 causes a distinct muscular dystrophy.

The extracellular matrix (ECM) has an important role in the development and maintenance of skeletal muscle, and several muscle diseases are associated with the dysfunction of ECM elements. MAMDC2 is a putative ECM protein and its role in cell proliferation has been investigated in certain cancer types. However, its participation in skeletal muscle physiology has not been previously studied. We describe 17 individuals with an autosomal dominant muscular dystrophy belonging to two unrelated families in which different heterozygous truncating variants in the last exon of MAMDC2 co-segregate correctly with the disease. The radiological aspect of muscle involvement resembles that of COL6 myopathies with fat replacement at the peripheral rim of vastii muscles. In this cohort, a subfascial and peri-tendinous pattern is observed in upper and lower limb muscles. Here we show that MAMDC2 is expressed in adult skeletal muscle and differentiating muscle cells, where it appears to localize to the sarcoplasm and myonuclei. In addition, we show it is secreted by myoblasts and differentiating myotubes into to the extracellular compartment. The last exon encodes a disordered region with a polar residue compositional bias loss of which likely induces a toxic effect of the mutant protein. The precise mechanisms by which the altered MAMDC2 proteins cause disease remains to be determined. MAMDC2 is a skeletal muscle disease-associated protein. Its role in muscle development and ECM-muscle communication remains to be fully elucidated. Screening of the last exon of MAMDC2 should be considered in patients presenting with autosomal dominant muscular dystrophy, particularly in those with a subfascial radiological pattern of muscle involvement.

A homozygous frameshift variant in SYCP2 caused meiotic arrest and non-obstructive azoospermia.

Genetic causation for the majority of non-obstructive azoospermia (NOA) remains unclear. Mutations in synaptonemal complex (SC)-associated genes could cause meiotic arrest and NOA. Previous studies showed that heterozygous truncating variants in SYCP2 encoding a protein essential for SC formation, are associated with non-obstructive azoospermia and severe oligozoospermia. Herein, we showed a homozygous loss-of-function variant in SYCP2 (c.2689_2690insT) in an NOA-affected patient. And this variant was inherited from heterozygous parental carriers by natural reproduction. HE, IF, and meiotic chromosomal spread analyses demonstrated that spermatogenesis was arrested at the zygotene stage in the proband with NOA. Thus, this study revealed that SYCP2 associated with NOA segregates in an autosomal recessive inheritance pattern, rather than an autosomal dominant pattern. Furthermore, our study expanded the knowledge of variants in SYCP2 and provided new insight into understanding the genetic etiology of NOA.

The genetic spectrum of congenital ocular motor apraxia type Cogan: an observational study, continued

BackgroundThe term congenital ocular motor apraxia (COMA), coined by Cogan in 1952, designates the incapacity to initiate voluntary eye movements performing rapid gaze shift, so called saccades. While regarded as a nosological entity by some authors, there is growing evidence that COMA designates merely a neurological symptom with etiologic heterogeneity. In 2016, we reported an observational study in a cohort of 21 patients diagnosed as having COMA. Thorough re-evaluation of the neuroimaging features of these 21 subjects revealed a previously not recognized molar tooth sign (MTS) in 11 of them, thus leading to a diagnostic reassignment as Joubert syndrome (JBTS). Specific MRI features in two further individuals indicated a Poretti–Boltshauser syndrome (PTBHS) and a tubulinopathy. In eight patients, a more precise diagnosis was not achieved. We pursued this cohort aiming at clarification of the definite genetic basis of COMA in each patient.ResultsUsing a candidate gene approach, molecular genetic panels or exome sequencing, we detected causative molecular genetic variants in 17 of 21 patients with COMA. In nine of those 11 subjects diagnosed with JBTS due to newly recognized MTS on neuroimaging, we found pathogenic mutations in five different genes known to be associated with JBTS, including KIAA0586, NPHP1, CC2D2A, MKS1, and TMEM67. In two individuals without MTS on MRI, pathogenic variants were detected in NPHP1 and KIAA0586, arriving at a diagnosis of JBTS type 4 and 23, respectively. Three patients carried heterozygous truncating variants in SUFU, representing the first description of a newly identified forme fruste of JBTS. The clinical diagnoses of PTBHS and tubulinopathy were confirmed by detection of causative variants in LAMA1 and TUBA1A, respectively. In one patient with normal MRI, biallelic pathogenic variants in ATM indicated variant ataxia telangiectasia. Exome sequencing failed to reveal causative genetic variants in the remaining four subjects, two of them with clear MTS on MRI.ConclusionsOur findings indicate marked etiologic heterogeneity in COMA with detection of causative mutations in 81% (17/21) in our cohort and nine different genes being affected, mostly genes associated with JBTS. We provide a diagnostic algorithm for COMA.

Spinocerebellar ataxia type 11 (SCA11): TTBK2 variants, functions and associated disease mechanisms.

Spinocerebellar ataxia type 11 (SCA11) is a rare type of autosomal dominant cerebellar ataxia, mainly characterized by progressive cerebellar ataxia, abnormal eye signs and dysarthria. SCA11 is caused by variants in TTBK2, which encodes tau tubulin kinase 2 (TTBK2) protein. Only a few families with SCA11 were described to date, all harbouring small deletions or insertions that result in frameshifts and truncated TTBK2 proteins. In addition, TTBK2 missense variants were also reported but they were either benign or still needed functional validation to ascertain their pathogenic potential in SCA11. The mechanisms behind cerebellar neurodegeneration mediated by TTBK2 pathogenic alleles are not clearly established. There is only one neuropathological report and a few functional studies in cell or animal models published to date. Moreover, it is still unclear whether the disease is caused by TTBK2 haploinsufficiency of by a dominant negative effect of TTBK2 truncated forms on the normal allele. Some studies point to a lack of kinase activity and mislocalization of mutated TTBK2, while others reported a disruption of normal TTBK2 function caused by SCA11 alleles, particularly during ciliogenesis. Although TTBK2 has a proven function in cilia formation, the phenotype caused by heterozygous TTBK2 truncating variants are not clearly typical of ciliopathies. Thus, other cellular mechanisms may explain the phenotype seen in SCA11. Neurotoxicity caused by impaired TTBK2 kinase activity against known neuronal targets, such as tau, TDP-43, neurotransmitter receptors or transporters, may contribute to neurodegeneration in SCA11.

Novel de novo ZNF148 truncating variant causing autism spectrum disorder, attention deficit hyperactivity disorder, and intellectual disability.

ZNF148 gene is a Krüppel-type transcription factor that has transcriptional regulatory function. Heterozygous variant in ZNF148 gene causes an intellectual disability syndrome characterized by global developmental delay, absence, or hypoplasia of corpus callosum, wide intracerebral ventricles, and dysmorphic facial features, while its associations with ASD and ADHD have not been reported. We report a new patient with intellectual disability, autism spectrum disorder (ASD) and attention-deficit hyperactivity disorder (ADHD). The patient had a novel heterozygous truncating variant c.1818dupC (p.Lys607Glnfs*11) in the ZNF148 gene. This variation produces a ZNF148 truncated protein with a deletion of the C-terminal activation domain and may destabilize the protein by affecting the transcriptional activation function. Brain MRI shows normal brain development. Here, we identify a novel ZNF148 heterozygous truncating variant in a patient with distinct phenotypes of ASD and ADHD, which expands the genotype-phenotype spectrum of ZNF148, and indicates ZNF148 is also a potential target gene for ASD.

PMON312 A De Novo Heterozygous Nonsense Variant In The SEC31A Gene Associated With Pituitary Hormone Deficiency And Disorders Of Sex Development.

Abstract Introduction XYdisorders of sex development (DSD) result from variants in many different human genes but frequently have no detectable molecular cause. In approximately 25% of cases of XY DSD, the index case may have associated malformations. Genetic disorders of endoplasmic reticulum (ER) function are increasingly being recognised but have not been associated with DSD or pituitary disorders. Clinical case Three siblings (with unaffected non-consanguineous parents) were reviewed at the tertiary endocrine clinic. Child I was noted at birth to have cliteromegaly. Imaging and examination under anaesthetic revealed a normal vagina and uterus but gonads of indeterminate origin. She was 46,XY and basal endocrine investigations at the age of 4 years showed a low AMH for male but otherwise normal gonadal and thyroid function and normal IGF-1. She had a laparoscopic bilateral gonadectomy aged 5 years. Pathology demonstrated bilateral testicular tissue, with substantial fibrotic atrophic change and occasional placental alkaline phosphatase (PLAP) positive cells, suggestive of germ cell tumours. Aged 8 years she developed obesity and later hypertension. Child II was reviewed due to short stature and diagnosed with GH deficiency aged 2 years. She has normal adrenal and thyroid function and gonadotrophins. MRI demonstrated an ectopic posterior pituitary. Child III presented with perineal hypospadias, a small phallus, bilateral undescended testes and craniofacial abnormalities. Endocrine investigations revealed hypogonadotrophic hypogonadism, with no testosterone response to hCG stimulation, a low normal AMH and no response of LH or FSH on LHRH stimulation. He has panhypopituitarism with an ectopic posterior pituitary gland on MRI and is currently on treatment with GH, hydrocortisone and levothyroxine. His BP is on the 98th centile for age and height. Child I and Child III have mild developmental delay but are in mainstream school with additional educational support. High-throughput DNA sequencing revealed, in all three siblings, a heterozygous truncating variant in the SEC31A gene that encodes a component of the COPII-complex that coats the vesicles mediating ER to Golgi transport. CRISPR-Cas9 targeted knockout of the corresponding Sec31a region resulted in embryonic lethality in homozygous mice. mRNA phenotyping of ER-related genes demonstrated increased mRNA expression of ATF4 and CHOP in the affected children, genes encoding key ER stress-related proteins, associated with defective protein transport. Conclusions Dysregulation ofanterograde and retrograde COPII-coated-vesicle ER-Golgi transport is increasingly recognised to underlie human developmental disorders, including Craniolenticulosutural dysplasia (OMIM 607812) and Saul-Wilson syndrome (OMIM 618150). The de novo SEC31A nonsense variant in all three affected siblings, the ER stress response, plus reported developmental syndromes with dysfunction of this transport mechanism and evidence from the preclinical mouse model suggest that SEC31A might underlie a previously unrecognised clinical syndrome comprising DSD, endocrine abnormalities, dysmorphic features and developmental delay. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

A Xp22.11-p21.3 microdeletion in a three-generation family supports male lethality of POLA1 nullisomy resulting in reduced fertility of female carriers

POLA1 encodes a subunit of the DNA polymerase alpha, a key enzyme for the initiation of DNA synthesis. In males, hemizygous hypomorphic variants in POLA1 have been identified as the cause of X-linked pigmentary reticulate disorder (XLPDR) and a novel X-linked neurodevelopmental disorder termed Van Esch-O’Driscoll syndrome (VEODS), while female carriers have been reported to be healthy. Nullisomy for POLA1 was speculated to be lethal due to its crucial function, while the effect of loss of one allele in females remained unknown. Here, we report on a three-generation family harboring a deletion of POLA1 in females showing subfertility as the only phenotype. Our findings show that heterozygous deletions or truncating variants in females with skewed X inactivation do not cause VEODS and support the hypothesis of very early embryonic lethality in males with POLA1 nullisomy.

Use of animal models to understand titin physiology and pathology.

In recent years, increasing attention has been paid to titin (TTN) and its mutations. Heterozygous TTN truncating variants (TTNtv) increase the risk of a cardiomyopathy. At the same time, TTNtv and few missense variants have been identified in patients with mainly recessive skeletal muscle diseases. The pathogenic mechanisms underlying titin‐related diseases are still partly unknown. Similarly, the titin mechanical and functional role in the muscle contraction are far from being exhaustively clarified. In the last few years, several animal models carrying variants in the titin gene have been developed and characterized to study the structural and mechanical properties of specific titin domains or to mimic patients' mutations. This review describes the main animal models so far characterized, including eight mice models and three fish models (Medaka and Zebrafish) and discusses the useful insights provided by a thorough characterization of the cell‐, tissue‐ and organism‐phenotypes in these models.

Two novel heterozygous truncating variants in NR4A2 identified in patients with neurodevelopmental disorder and brief literature review.

Pathogenic variants in the nuclear receptor superfamily 4 group A member 2 (NR4A2) cause an autosomal dominant neurodevelopmental disorder with or without seizures. Here, we described two patients presenting with developmental delay, language impairment, and attention-deficit hyperactivity disorder. Trio-based whole exome sequencing revealed two novel heterozygous variants, c.1541-2A > C and c.915C > A, in NR4A2. Both variants were identified as de novo and confirmed by Sanger sequencing. In vitro functional analyses were performed to assess their effects on expression of mRNA or protein. The canonical splicing variant c.1541-2A > C caused aberrant splicing, leading to the retention of intron 7 and a truncated protein due to an early termination codon within intron 7 with decreased protein expression, while the variant c.915C > A was shown to result in a shorter protein with increased expression level unexpectedly. The clinical and genetic characteristics of the previously published patients were briefly reviewed for highlighting the potential link between mutations and phenotypes. Our research further confirms that NR4A2 is a disease-causing gene of neurodevelopmental disorders and suggests alterations in different domains of NR4A2 cause various severity of symptoms.

The Involvement of ALPK3 in Hypertrophic Cardiomyopathy in East Asia.

ObjectiveALPK3 is associated with a recessive form of pediatric cardiomyopathy accompanied by musculoskeletal and craniofacial abnormalities. Heterozygous truncating variants in this gene (ALPK3tv) have recently been confirmed as a cause of autosomal dominant hypertrophic cardiomyopathy (HCM). Whether ALPK3 is also implicated in HCM in East Asia and the effect of missense variants in ALPK3 on HCM remains unresolved.MethodsWe compared the frequency of rare deleterious variants in ALPK3 in a study cohort comprised of 793 HCM cases of East Asian descent to that in the controls subset of Genome Aggregation Database (gnomAD). Gene burden test was used to assess this association. The involvement of these variants in HCM was further validated by independent cohort. The clinical characteristics and prognoses of these carriers were compared with sarcomere-positive and negative patients.ResultsRare deleterious variants in ALPK3 were significantly enriched in HCM compared with gnomAD controls (truncating: 4/793 vs. 4/4523, P = 0.02; missense: 25/793 vs. 46/4523, P = 2.56e-5). Replication in an independent cohort provided more supporting evidence. Further comparisons revealed that ALPK3 carriers displayed more severe hypertrophy in interventricular septum (IVS) and apex, as well as greater maximal left ventricular wall thickness, relative to sarcomere negatives.ConclusionHeterozygous rare variants in ALPK3, both missense and truncating variants, are associated with HCM in East Asians.

Autosomal Dominant Retinitis Pigmentosa-Associated TOPORS Protein Truncating Variants Are Exclusively Located in the Region of Amino Acid Residues 807 to 867.

PurposeHeterozygous truncating variants of TOPORS have been reported to cause autosomal dominant retinitis pigmentosa (adRP). The purpose of this study was to investigate whether all heterozygous truncating variants, including copy number variants (CNVs), are pathogenic.Methods TOPORS truncating variants were collected and reviewed through an in-house dataset and existing databases. Individuals with truncating variants underwent ophthalmological evaluation.ResultsSix truncating variants were detected in seven families. Three N-terminus truncating variants were detected in three families without RP, and the other three were identified in four unrelated families with typical RP. Based on the in-house dataset and published literature, 17 truncating variants were identified in 47 families with RP. All RP-associated truncating alleles, except one, were distributed in the last exon of TOPORS and clustered in amino acid residues 807 to 867 (46/47, 97.9%). Conversely, in the gnomAD database, only one truncating allele (1/27, 3.7%) was in this region, and the others were outside (26/27, 96.3%), suggesting that the pathogenic truncating variants were significantly clustered in residues 807 to 867 (χ2 = 65.6, P = 1.1 × 10–17). Additionally, three CNVs involving the N-terminus of TOPORS were recorded in control populations but were absent in affected patients.ConclusionsThis study suggests that all pathogenic truncating variants of TOPORS were clustered in residues 807 to 867, whereas the truncating variants outside this region and the CNVs involving the N-terminus were not associated with RP. A dominant-negative effect, rather than haploinsufficiency, is speculated to be the underlying pathogenesis. These findings provide valuable information for interpreting variation in TOPORS and other genes in similar situations, especially for CNVs.

Neurofibromatosis Type 1 Has a Wide Spectrum of Growth Hormone Excess.

Overgrowth due to growth hormone (GH) excess affects approximately 10% of patients with neurofibromatosis type 1 (NF1) and optic pathway glioma (OPG). Our aim is to describe the clinical, biochemical, pathological, and genetic features of GH excess in a retrospective case series of 10 children and adults with NF1 referred to a tertiary care clinical research center. Six children (median age = 4 years, range of 3–5 years), one 14-year-old adolescent, and three adults (median age = 42 years, range of 29–52 years) were diagnosed with NF1 and GH excess. GH excess was confirmed by the failure to suppress GH (<1 ng/mL) on oral glucose tolerance test (OGTT, n = 9) and frequent overnight sampling of GH levels (n = 6). Genetic testing was ascertained through targeted or whole-exome sequencing (n = 9). Five patients (all children) had an OPG without any pituitary abnormality, three patients (one adolescent and two adults) had a pituitary lesion (two tumors, one suggestive hyperplasia) without an OPG, and two patients (one child and one adult) had a pituitary lesion (a pituitary tumor and suggestive hyperplasia, respectively) with a concomitant OPG. The serial overnight sampling of GH levels in six patients revealed abnormal overnight GH profiling. Two adult patients had a voluminous pituitary gland on pituitary imaging. One pituitary tumor from an adolescent patient who harbored a germline heterozygous p.Gln514Pro NF1 variant stained positive for GH and prolactin. One child who harbored a heterozygous truncating variant in exon 46 of NF1 had an OPG that, when compared to normal optic nerves, stained strongly for GPR101, an orphan G protein-coupled receptor causing GH excess in X-linked acrogigantism. We describe a series of patients with GH excess and NF1. Our findings show the variability in patterns of serial overnight GH secretion, somatotroph tumor or hyperplasia in some cases of NF1 and GH excess. Further studies are required to ascertain the link between NF1, GH excess and GPR101, which may aid in the characterization of the molecular underpinning of GH excess in NF1.

Abstract 12802: Intron-Mediated Enhancement of Titin ( TTN ) Regulates Sarcomere Formation and Function

Background: Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure, a major cause of morbidity and mortality. This causality indicates that even two-fold changes in the amount of TTN can profoundly disturb cardiac physiology. Although a critical role of TTN in sarcomere formation and cardiomyocyte contractility is well established, the mechanisms regulating transcription of the TTN gene remain poorly understood. Methods: We performed bioinformatics analysis to identify a putative transcriptional enhancer of TTN . Next, we created biallelic deletion of the enhancer in human induced pluripotent stem cell derived cardiomyocytes and performed enhancer reporter assays both in vitro and in vivo to demonstrate necessity and sufficiency of the enhancer in TTN gene expression, respectively. Furthermore, we performed massive parallel reporter assay to define critical transcriptional factors of the TTN enhancer activity and analyzed whole genome sequencing (WGS) data of human patients with unexplained dilated cardiomyopathy (DCM). Results: We identified an intron mediated enhancer that promotes cardiac-specific TTN expression. Global deletion of this element downregulated TTN expression in cardiomyocytes and impaired sarcomere development, while transgenic expression promoted cardiac expression in mice. Using mutational scanning we defined key transcription factor binding sites, including NKX2-5 and MEF2 motifs that promote TTN expression in cardiomyocytes. Consistent with these functions, analyses of WGS data in 69 patients with unexplained DCM revealed one rare variant that disrupted the conserved MEF2 transcriptional factor binding motif. Conclusions: Discovery of a TTN enhancer advances our understanding of cardiomyocyte development, provides an opportunity to modulate TTN transcriptional activity, and ultimately develop therapeutic strategies to treat dilated cardiomyopathy caused by TTN haploinsufficiency.

Abstract 10240: Key Pathomechanisms of Cardiomyopathy Due to TTN Truncation

Introduction: Heterozygous truncating variants in TTN (TTNtv) coding for titin cause dilated cardiomyopathy (DCM) but the underlying pathomechanisms are unclear and disease management remains uncertain. Here, we aim to elucidate the key pathomechanisms of TTNtv-DCM. Hypothesis: Stable expression of truncated titin proteins and titin haploinsufficiency characterize TTNtv-DCM and represent potential targets for therapy. Methods and Results: We studied left ventricular tissues from 14 nonfailing donor hearts and 113 endstage failing DCM patients and identified a TTNtv in 22 DCM patients (19.5%) by next generation sequencing. Using titin protein gel electrophoresis and western blot we demonstrate, for the first time, titin haploinsufficiency in TTNtv-DCM hearts. Strikingly, all 21 adult TTNtv-DCM hearts of our cohort showed stable expression of truncated titin proteins. Expression was variable, up to one-half of the total titin protein pool, and negatively correlated to patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates. Deregulated ubiquitin-dependent protein quality control was apparent. Next, we produced human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), comparing wildtype controls to cells with a patient-derived, prototypical A-band TTNtv or a CRISPR/Cas9-edited M-band TTNtv. TTNtv hiPSC-CMs showed reduced wildtype titin expression and contained truncated titin proteins, whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR/Cas9, thereby eliminating truncated titin proteins and raising wildtype titin content. Functional improvement also occurred when titin protein content was increased by proteasome inhibition. Conclusions: Our study reveals the major pathomechanisms of TTNtv-DCM, which include titin haploinsufficiency as a life-long condition and truncated titin protein enrichment with aggregate formation, along with aberrant protein quality control. Results can be exploited for new therapies of TTNtv cardiomyopathies.