Heterozygous Sequence Variants Research Articles

Unveiling genetic insights: Array-CGH and WES discoveries in a cohort of 122 children with essential autism spectrum disorder

BackgroundAutistic Spectrum Disorder (ASD) is a neurodevelopmental disorder with a strong genetic component and high heterogeneity. Essential ASD refers to patients who do not have other comorbidities. This study aimed to investigate the genetic basis of essential ASD using whole exome sequencing (WES) and array-comparative genomic hybridization (array-CGH).ResultsIn a cohort of 122 children with essential ASD, WES detected 382 variants across 223 genes, while array-CGH identified 46 copy number variants (CNVs). The combined use of WES and array-CGH revealed pathogenic variants in four patients (3.1% detection rate) and likely pathogenic variants in 34 patients (27.8% detection rate). Only one patient had a pathogenic CNV (0.8% detection rate). Including likely pathogenic variants, the overall detection rate was 31.2%. Additionally, 33 de novo heterozygous sequence variants were identified by WES, with three classified as pathogenic and 13 as likely pathogenic. Sequence variants were found in 85 genes already associated with ASD, and 138 genes not previously included in the SFARI dataset were identified as potential new candidate genes.ConclusionsThe study enhances genetic understanding of essential ASD and identifies new candidate genes of interest. The findings suggest that using both array-CGH and WES in patients with essential ASD can improve the detection of pathogenic and likely pathogenic genetic variants, contributing to better diagnosis and potentially guiding future research and treatment strategies.

7039 Isolated Gonadotropin Deficiency Transmitted From Mother to Daughter Conceived Through Assisted Reproduction Due Newly Identified, Autosomal Dominant Mutation Within the SOX10 Gene

Abstract Disclosure: S. Bakjaji: None. R.P. Hoffman: None. Title: Isolated gonadotropin deficiency transmitted from mother to daughter conceived through assisted reproduction due newly identified, autosomal dominant mutation within the SOX10 gene. Objectives: We report a case of genetic isolated gonadotropin deficiency occurring in a mother and in her daughter conceived through assisted reproductive technology using mother’s ovum. Methods Case report Results: The daughter was initially seen at 12 years 0 months for evaluation of short stature and concerns she had not grown in the last year. She was otherwise in good health and reported normal sense of smell. At the time of assessment, her height was 132.6 cm (0.61 percentile; Z=-2.5). The mother had been diagnosed with Kallmann’s syndrome and achieved pregnancy through ovarian stimulation. Her height was 152.4 cm, while the father's height was 177.8 cm. Two male siblings were born at the same time and were in normal health. Physical examination of the daughter was unremarkable. She had Tanner stage 1 pubic hair and Tanner stage 1 breasts. The growth chart displayed a notable deceleration in growth Laboratory results showed normal free T4 of (0.9 ng/dL) with slightly elevated TSH (8.348 uIU/mL), positive thyroid antibodies, normal IGFI at 164 ng/mL (normal range for Tanner I: 54 - 301) and IGFBP-3 at 5.8 ug/mL (2.4 - 8.4 ug/mL). The karyotype was 46 XX. Her peak growth hormone using glucagon-arginine stimulation was 12.8 ng/ml. When no pubertal development was seen over the next 6 months, GnRH stimulation testing revealed baseline LH of 0.04 mIU/ml and a peak of 1.78 and baseline FSH of 0.41 mIU/ml with a peak of 2.83. Bone age was 8 years 4 months.Over the next year her growth velocity was 4.8 cm/year and she remained prepubertal. Genetic testing done identified a new heterozygous sequence variant (c.226G>A) in the SOX10 gene which would lead to replacement of valine by isoleucine at position 76 and was thought to be damaging in silico. This variant was subsequently found to be shared with her mother but not her father. Conclusions: Most Kallmann’s syndrome or genetic isolated gonadotropin deficiency cases are due to autosomal dominant but spontaneous new mutations since infertility is usually present in those with the syndrome. This case emphasizes the importance of genetic counselling regarding inheritance risk when assisted reproduction technology using maternal ovum is used. Presentation: 6/2/2024

Identifying subgroups of childhood obesity by using multiplatform metabotyping.

Introduction: Obesity results from an interplay between genetic predisposition and environmental factors such as diet, physical activity, culture, and socioeconomic status. Personalized treatments for obesity would be optimal, thus necessitating the identification of individual characteristics to improve the effectiveness of therapies. For example, genetic impairment of the leptin-melanocortin pathway can result in rare cases of severe early-onset obesity. Metabolomics has the potential to distinguish between a healthy and obese status; however, differentiating subsets of individuals within the obesity spectrum remains challenging. Factor analysis can integrate patient features from diverse sources, allowing an accurate subclassification of individuals. Methods: This study presents a workflow to identify metabotypes, particularly when routine clinical studies fail in patient categorization. 110 children with obesity (BMI > +2 SDS) genotyped for nine genes involved in the leptin-melanocortin pathway (CPE, MC3R, MC4R, MRAP2, NCOA1, PCSK1, POMC, SH2B1, and SIM1) and two glutamate receptor genes (GRM7 and GRIK1) were studied; 55 harboring heterozygous rare sequence variants and 55 with no variants. Anthropometric and routine clinical laboratory data were collected, and serum samples processed for untargeted metabolomic analysis using GC-q-MS and CE-TOF-MS and reversed-phase U(H)PLC-QTOF-MS/MS in positive and negative ionization modes. Following signal processing and multialignment, multivariate and univariate statistical analyses were applied to evaluate the genetic trait association with metabolomics data and clinical and routine laboratory features. Results and Discussion: Neither the presence of a heterozygous rare sequence variant nor clinical/routine laboratory features determined subgroups in the metabolomics data. To identify metabolomic subtypes, we applied Factor Analysis, by constructing a composite matrix from the five analytical platforms. Six factors were discovered and three different metabotypes. Subtle but neat differences in the circulating lipids, as well as in insulin sensitivity could be established, which opens the possibility to personalize the treatment according to the patients categorization into such obesity subtypes. Metabotyping in clinical contexts poses challenges due to the influence of various uncontrolled variables on metabolic phenotypes. However, this strategy reveals the potential to identify subsets of patients with similar clinical diagnoses but different metabolic conditions. This approach underscores the broader applicability of Factor Analysis in metabotyping across diverse clinical scenarios.

Targeting oncogenic TERT promoter variants by allele-specific epigenome editing

BackgroundActivation of dominant oncogenes by small or structural genomic alterations is a common driver mechanism in many cancers. Silencing of such dominantly activated oncogenic alleles, thus, is a promising strategy to treat cancer. Recently, allele-specific epigenome editing (ASEE) has been described as a means to reduce transcription of genes in an allele-specific manner. In cancer, specificity to an oncogenic allele can be reached by either targeting directly a pathogenic single-nucleotide variant or a polymorphic single-nucleotide variant linked to the oncogenic allele. To investigate the potential of ASEE in cancer, we here explored this approach by targeting variants at the TERT promoter region. The TERT promoter region has been described as one of the most frequently mutated non-coding cancer drivers.ResultsSequencing of the TERT promoter in cancer cell lines showed 53% (41/77) to contain at least one heterozygous sequence variant allowing allele distinction. We chose the hepatoblastoma cell line Hep-G2 and the lung cancer cell line A-549 for this proof-of-principle study, as they contained two different kinds of variants, namely the activating mutation C228T in the TERT core promoter and the common SNP rs2853669 in the THOR region, respectively. These variants were targeted in an allele-specific manner using sgRNA-guided dCas9-DNMT3A-3L complexes. In both cell lines, we successfully introduced DNA methylation specifically to the on-target allele of the TERT promoter with limited background methylation on the off-target allele or an off-target locus (VEGFA), respectively. We observed a maximum CpG methylation gain of 39% and 76% on the target allele when targeting the activating mutation and the common SNP, respectively. The epigenome editing translated into reduced TERT RNA expression in Hep-G2.ConclusionsWe applied an ASEE-mediated approach to silence TERT allele specifically. Our results show that the concept of dominant oncogene inactivation by allele-specific epigenome editing can be successfully translated into cancer models. This new strategy may have important advantages in comparison with existing therapeutic approaches, e.g., targeting telomerase, especially with regard to reducing adverse side effects.

The grape powdery mildew resistance loci Ren2, Ren3, Ren4D, Ren4U, Run1, Run1.2b, Run2.1, and Run2.2 activate different transcriptional responses to Erysiphe necator.

Multiple grape powdery mildew (PM) genetic resistance (R) loci have been found in wild grape species. Little is known about the defense responses associated with each R locus. In this study, we compare the defense mechanisms associated with PM resistance in interspecific crosses segregating for a single R locus from Muscadinia rotundifolia (Run1, Run1.2b, Run2.1, Run2.2), Vitis cinerea (Ren2), V. romanetii (Ren4D and Ren4U), and the interspecific hybrid Villard blanc (Ren3). By combining optical microscopy, visual scoring, and biomass estimation, we show that the eight R loci confer resistance by limiting infection at different stages. We assessed the defense mechanisms triggered in response to PM at 1 and 5 days post-inoculation (dpi) via RNA sequencing. To account for the genetic differences between species, we developed for each accession a diploid synthetic reference transcriptome by incorporating into the PN40024 reference homozygous and heterozygous sequence variants and de novo assembled transcripts. Most of the R loci exhibited a higher number of differentially expressed genes (DEGs) associated with PM resistance at 1 dpi compared to 5 dpi, suggesting that PM resistance is mostly associated with an early transcriptional reprogramming. Comparison of the PM resistance-associated DEGs showed a limited overlap between pairs of R loci, and nearly half of the DEGs were specific to a single R locus. The largest overlap of PM resistance-associated DEGs was found between Ren3 +, Ren4D +, and Ren4U + genotypes at 1 dpi, and between Ren4U + and Run1 + accessions at 5 dpi. The Ren3 +, Ren4D +, and Ren4U + were also found to have the highest number of R locus-specific DEGs in response to PM. Both shared and R locus-specific DEGs included genes from different defense-related categories, indicating that the presence of E. necator triggered distinct transcriptional responses in the eight R loci.

RF14 | PSAT120 X-Linked Central Precocious Puberty Associated with MECP2 Defects

Abstract Methyl-CpG-binding protein 2 (MECP2) is a chromatin-associated protein that can both activate and repress transcription, playing an essential role in neuronal maturation. MECP2 is encoded by an X-linked gene (chromosome Xq28) with high expression in the brain, including the hypothalamic region. Loss-of-function mutations in MECP2 are usually associated with Rett syndrome, a rare genetic disorder characterized by normal early development followed by regression of acquired skills, such as purposeful hand movements and the ability to communicate, repetitive hand stereotypies, slowed brain growth, seizures, and intellectual disability with female predominance. Notably, early pubertal development (early thelarche, pubarche and, less often, menarche) has been demonstrated in girls with Rett syndrome. In this original study, a total of 329 CPP patients (308 girls and 21 boys) were investigated for MECP2 defects. Familial cases were identified in 38% of this CPP cohort. First, 129 CPP patients were enrolled for multigene sequencing studies (whole-exome sequencing n=58; targeted gene sequencing n=71) as part of genetic investigations based on large-scale approaches. Three rare heterozygous sequence variants predicted to be deleterious in MECP2 gene were identified in 5 girls from 4 unrelated families with CPP: the p.Arg97Cys de novo missense variant in two monozygotic twin sisters with CPP and microcephaly; the p.Ser176Arg de novo missense variant in one girl with sporadic CPP, obesity and autism; and the p.Ala6_Ala8dup insertion in two unrelated girls with sporadic CPP (one with a maternally inherited variant). To expand the investigation for potentially damaging sequence variants of MECP2, a larger multiethnic cohort of 200 CPP patients was further analyzed through Sanger sequencing. One rare heterozygous 3'UTR insertion in MECP2 (c.*36_*37insT) was identified in two unrelated girls with sporadic CPP (one with a maternally inherited variant and other with a de novo variant). None of these MECP2 variants have been previously reported in Rett syndrome cases. The girls with CPP associated with MECP2 defects did not manifest typical Rett syndrome. The three patients with more deleterious MECP2 variants, according to in silico prediction tools, had neurobehavioral phenotypes, likely corresponding to a genotype-phenotype correlation. Immunohistochemistry and immunofluorescence studies were performed in pubertal female mice. Abundant staining for Mecp2 was demonstrated in multiple hypothalamic nuclei (arcuate, suprachiasmatic, and paraventricular) and in the median eminence. Co-localization of Mecp2 and GnRH within GnRH neurons was suggested in double labelling experiments. Hence, Mecp2 expression was demonstrated in key hypothalamic nuclei responsible for GnRH regulation in female mice. In conclusion, we have identified rare dominant MECP2 defects in multiple unrelated girls with CPP with or without mild neurodevelopmental abnormalities, revealing a new X-linked form of premature reactivation of the hypothalamic-pituitary-gonadal axis. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 12:30 p.m. - 12:35 p.m.

A de novo YY1 missense variant expanding the Gabriele-de Vries syndrome phenotype and affecting X-chromosome inactivation.

Yin and Yang 1 gene (YY1; MIM#600,013) is recognized as a dual transcriptional activating and repressing factor, RNA-binding protein, and 3D chromatin regulator, with multi roles in neurodevelopmental and maintenance pathways. YY1 haploinsufficiency caused either by heterozygous sequence variants or deletions involving the whole gene has been recently associated with Gabriele-de Vries syndrome (GADEVS), a rare congenital autosomal dominant condition, leading to intellectual disability (ID) and multiple physical/behavioural abnormalities. Herein, we describe clinical and molecular findings from a Brazilian female harbouring a de novo missense pathogenic variant in YY1 gene (NM_003403.5:c.1106A > G; p.Asn369Ser) found by whole exome sequencing with potential implications for protein structure and function. Undescribed or uncommon clinical features in this patient included non-febrile seizures, severe scoliosis, hearing impairment, and chorioretinitis. Further bioinformatics analyses using YY1-other protein interaction networks reinforced the involvement of YY1 interactors in such phenotypes, in exception of chorioretinitis. Moreover, X-chromosome inactivation (XCI) skewing was evidenced in the patient and attributed to the haploinsufficiency of YY1, which direct and indirectly interacts with numerous XCI key regulators. Besides expanding the mutational and phenotype spectrum of GADEVS, our results highlight the role of YY1 as an essential autosomal regulator of XCI epigenetic process.

O’Donnell-Luria-Rodan syndrome: description of a second multinational cohort and refinement of the phenotypic spectrum

BackgroundO’Donnell-Luria-Rodan syndrome (ODLURO) is an autosomal-dominant neurodevelopmental disorder caused by pathogenic, mostly truncating variants in KMT2E. It was first described by O’Donnell-Luria et al in 2019 in a cohort of...

EP200: Noonan syndrome associated with focal occipital alopecia in a patient with RAF1 variant: A case report and literature review

Noonan syndrome (NS) is a heterogenous condition characterized by distinctive craniofacial features, cardiac defects, and neurodevelopmental changes and is caused by variants in one of seven genes coding for proteins within the Ras-MAPK pathway. Pathogenic variants in RAF1 account for approximately 3-17% of cases and are associated with a high prevalence of severe hypertrophic cardiomyopathy (HCM) and pulmonary hypertension (PHTN). Cerebrovascular and Chiari I malformations, although rare, have also been described in individuals with RAF-1 NS. To our knowledge, the presence of congenital occipital alopecia in an individual with NS has not been previously reported. We report a patient with a de novo pathogenic heterozygous RAF1 gene sequence variant, c.770C>T (p.Ser257Leu), with atypical scalp findings. Our proband was a 4-week-old female dichorionic, diamniotic twin born at 35 weeks 2 days gestation to nonconsanguineous parents. At birth, her length was 45.7 cm (14th centile), weight was 2656 g (69th centile), and head circumference was 34.5 cm (97th centile). The mother was 38 at the time of birth. There was no history of maternal or gestational diabetes. Prenatal ultrasound was concerning for cystic hygroma. The immediate neonatal course was complicated by acute respiratory failure, left ventricular outflow tract obstruction with concern for coarctation on echocardiogram, poor feeding, and failed newborn hearing screen which prompted transfer to a tertiary NICU for further management. At the time of consultation, the following problems had been identified: bilateral cataracts, significant redundant posterior neck folds that showed fatty soft tissue neck mass without a cystic component, and hypertrophic cardiomyopathy with elevated pulmonary arterial pressures. Physical exam was notable for relative macrocephaly with turricephaly, lowset, posteriorly rotated ears, redundant posterior neck folds, and a focal, 1cm circular area of alopecia at the midline occipital region. These findings were concerning for a RASopathy. Imaging of her superficial midline scalp defect with brain MRI showed a suspected atretic encephalocele, but CT did not reveal any occipital bony defects. After re-evaluation, it was confirmed that there was no intracranial connection or bony defect at the area of the congenital alopecia nor at the level of the suboccipital skin thickening/redundancy. Chromosomal microarray was nondiagnostic and thus exome sequence analysis was preformed which detected the pathogenic RAF1 variant. The diagnosis of RAF1 NS accounts for nearly all of the physical changes seen in our patient, particularly the severe degree of PHTN and HCM, with the exception of her occipital alopecia. Since our initial consultation, the patient continues to grow along NS-specific curve, has persistent mild bilateral hearing loss, and has had interval placement of gastric tube secondary to oral feeding difficulties. There has been mild improvement in cardiac function, septal hypertrophy, and PHTN. She remains seizure-free. This report expands the phenotype of RAF1 Noonan syndrome to include congenital focal alopecia. We recommend that NS should be added to the differential when this same scalp finding is present. Although the presence of this physical finding in our patient was not definitively associated with any underlying structural CNS or bony changes, this case highlights the importance of investigating with detailed cranial and spinal imaging to better characterize cerebral morphology and guide genetic testing in suspected RASopathy cases. Additional cases with this finding are needed to better understand potential associations.

ASH1L mutation caused seizures and intellectual disability in twin sisters

ASH1L mutations have been identified with variable phenotypes, including intellectual disability, autism spectrum disorder (ASD), and multiple congenital anomalies (MCA). However, the mechanisms underlying this phenotypic variation remain unknown. Here, we present twin sisters exhibiting mild intellectual disability and seizures. Whole-exome sequencing of the family revealed a novel de novo heterozygous sequence variant, NM_018489.2: c.2678dup (p.Lys894*) in exon 3 of ASH1L which was estimated to be pathogenic. Furthermore, we reviewed previously reported ASH1L mutations in order to evaluate genotype-phenotype correlations for ASH1L variants. We found that patients with missense mutations in ASH1L appeared to present with more severe phenotypes and a higher likelihood of ASD than those with truncating mutations. The relationship between phenotype and genotype reported across several patients may help to explain the mechanisms underlying the phenotypic variation commonly observed between ASH1L mutations.

Pro-Platelet Formation Is Impaired in Megakaryocytes of MYH9 -Related Disease Patients

IntroductionThe MYH9 gene encodes myosin-IIA, a nonmuscle myosin heavy chain that facilitates shape changes in diverse cell types. The mutation of MYH9 may lead to the MYH9 -related disease (MYH9 -RD), an autosomal dominant disorder with macrothrombocytopenia and leukocyte inclusion bodies at birth and the risk of developing nephropathy, deafness and cataracts during infancy or adult life. Currently, at least 44 different MYH9 mutations in all 40 exons have been reported. Mutated MYH9 protein expression is reduced in megakaryocytes and is clumped in a few aggregates together with the wild-type protein in neutrophils, where they form the inclusion bodies. In a mouse model, myosin-IIA protein inhibits pro-platelet formation and is regulated by Rho-associated kinase (ROCK). However, how the mutated myosin-IIA influences the pro-platelet formation (PPF) of megakaryocytes in patients has not been explored. The aim of this study is to determine the function and regulation of mutated myosin-IIA in MYH9 -RD patients' megakaryocytes.MethodsOne kindred with inherited macrothrombocytopenia was identified and 5 healthy donors were enrolled as controls. Peripheral blood smear review, slit lamp examination, pure-tone audiometry, renal ultrasonography and examination of blood (urea, creatinine, electrolytes) and urine were performed for each family member to diagnose and evaluate the disease. Bidirectional polymerase chain reaction (PCR) amplification and DNA sequence analysis were performed on all 40 MYH9 exons for each patient. Platelets from peripheral blood and megakaryocytes from bone marrow were fractionated for electron microscopy. Bone marrow tissues from patients were stained with fluorescein-labeled antibodies against CD41 and CD144 to observe the distribution of mature megakaryocytes in the vascular niche by confocal fluorescence microscopy. CD34+ cells were isolated from bone marrow samples by cell sorting for in vitro culture with TPO. On day 8, megakaryocytes were enriched using an albumin density gradient and seeded onto 96-well plates in serum-free medium with or without Y27632, the ROCK inhibitor. At the same time every day for 5 days, the percentage of proplatelet-displaying megakaryocytes was quantified by inverted microscopy.ResultsFive patients in the kindred were diagnosed with MYH9 -RD based upon peripheral blood smear review, one of whom had severe renal failure. None of the patients suffered from deafness or cataracts. A pathogenic heterozygous sequence variant in exon 39 (c.5521G>A, p.E1841K) was detected, which substitutes glutamate at codon 1841 to lysine. This mutation is one of the 44 reported pathogenic mutations. Electron microscopic results showed that platelets from patients had increased diameters and irregular shapes while megakaryocytes from patients had smaller diameters and a disorganized demarcation membrane system (DMS). Immunofluorescence results of bone marrow tissues showed that proplatelet-displaying mature megakaryocytes in MYH9 -RD patients located distally to the vascular niche, compared with the control group. CD34+ cells from bone marrow differentiated into megakaryocytes after in vitro culture with TPO. However, the PPF of megakaryocytes from MYH9 -RD patients appeared much earlier than that from controls. At the same time point, PPF megakaryocytes in MYH9 -RD patients were significantly increased compared to those of the control group. When cultured with Y27632, the PPF appearance time and proplatelet-displaying megakaryocyte percentage in the control group were advanced, while the MYH9 -RD group was not influenced.ConclusionThe mutation in exon 39 of the MYH9 gene may lead to morphological abnormalities of megakaryocytes and platelets. The regulation of myosin-IIA function by ROCK in patients may be decreased when the MYH9 gene is mutated. As a result, the timing of PPF of megakaryocytes with mutated myosin-IIA in the bone marrow niche of MYH9 -RD patients may be advanced, and their mis-location away from the vascular niche may lead to reduced platelet production and increased platelet destruction. DisclosuresNo relevant conflicts of interest to declare.

Lymphedema distichiasis syndrome may be caused by FOXC2 promoter-enhancer dissociation and disruption of a topological associated domain.

Lymphedema distichiasis syndrome (LDS) is a rare autosomal dominant condition characterized by lower limb lymphedema, distichiasis, and variable additional features. LDS is usually caused by heterozygous sequence variants in the FOXC2 gene located at 16q24, but in one previous instance LDS has resulted from a balanced reciprocal translocation with a breakpoint at 16q24, 120 kb distal to the FOXC2 gene suggesting a position effect. Here, we describe a second family with LDS caused by a translocation involving 16q24. The family were ascertained after detection of a paternally inherited balanced reciprocal translocation t(16;22)(q24;q13.1) in a pregnancy complicated by severe fetal hydrops. There was a past history of multiple miscarriages in the father's family, and a personal and family history of lymphedema and distichiasis, consistent with the diagnosis of LDS. Using whole genome amplified DNA from single sperm of the male proband, bead array analysis demonstrated that the FOXC2 gene was intact and the chromosome 16 breakpoint mapped to the same region 120Kb distal to the FOXC2 gene. This case highlights the clinical consequences that can arise from a translocation of genomic material without dosage imbalance, and that it is increasingly feasible to predict and characterize possible effects with improved access to molecular techniques.

348 A case of phaeohyphomycosis caused by Corynespora cassiicola, a plant pathogen

Corynespora cassiicola is a common plant pathogen. Humans infected by C. cassiicola are extremely rare. We report a case of subcutaneous and pulmonary phaeohyphomycosis caused by C. cassiicola in a 68-year-old male Chinese. The clinical manifestations were ulcers on the upper arm skin with erosions, scabs, and infiltrative plaques. The pulmonary manifestations presented with cough, chest tightness, and fever. The organism was identified as C. cassiicola based on the morphology and the sequence of the internal transcribed spacer region of the ribosomal RNA gene. CARD9 deficiency has been reported to underlie several fungal infections. By employing whole-exome capture, compound heterozygous sequence variants were found in the CARD9 gene, c.106C>T (p.Gln36*) and c.1118G>C (p.Arg373Pro). The patient was cured by the antifungal drug voriconazole, based on repeated fungal microscopy and culture which were negative. The skin lesions gradually healed, leaving scar and erosive surface. There are less than 10 reports on subcutaneous infection by C. cassiicola, and this case is unique with concomitant subcutaneous and pulmonary phaeohyphomycosis highlighting the role of CARD9 in the human immune response in controlling fungal infections. In summary, this case emphasizes the importance of genetic susceptibility to certain microbial infections.

1801-P: Novel De Novo INS P.T97P Variant Presenting with Severe Neonatal DKA

Objective: A 5 month old, premature male infant with history of hydrops fetalis and hernia repair presented unresponsive in respiratory distress requiring intubation and was found to have pH 6.93, bicarbonate 6 mmol/L, anion gap 23 mmol/L, glucose 669 mg/dL and HbA1c 14.2%. His condition improved with insulin drip, fluid and electrolyte management. Work-up revealed negative autoantibodies and the University of Chicago Neonatal Diabetes Sequencing Panel was performed. Testing revealed a heterozygous sequence variant of unknown significance in the INS gene (c.289A>C), resulting in the amino acid change p.T97P. In silico pathogenicity prediction tools provided contradictory results. Neither parent carried the variant. INS gene mutations have been associated with Mutant INS-gene-induced Diabetes of Youth (MIDY) that includes permanent neonatal diabetes and MODY 10. Expression of mutant proinsulin is thought to lead to misfolded complexes in the endoplasmic reticulum (ER), leading to ER stress and β-cell death. Methods: To determine the effects of this variant on insulin processing and release, we utilized a validated luminescent insulin secretion assay where Gaussia luciferase is inserted into the C-peptide portion of proinsulin. We compared luciferase secretion for wild type and variant INS sequences. Results: The mutation decreased luciferase release ∼10-fold versus wild type. Proteasome inhibition led to partial rescue. However, overall expression of proinsulin-luciferase peptide was unchanged between the wild type and mutant. Therefore, it is likely that the mutation causes disruptions in processes downstream of transcription and translation, such as folding, proteolytic processing and transport. Conclusions: The novel INS p.T97P is the first identified A-chain mutation resulting in MIDY not involving the creation or deletion of cysteine residues. The substitution of the polar threonine with nonpolar proline may be sufficient to disrupt nearby disulfide bonding in the proinsulin folding pathway. Disclosure R. Lal: Consultant; Self; Abbott, Biolinq. H.P. Moeller: None. R. Freeman: None. P. Prahalad: None. J.P. Annes: None. Funding National Institutes of Health (1K12DK122550, T32DK007217, R01DK101530, T32GM113854, P30DK116074)

Expanding the phenotype of PURA-related neurodevelopmental disorder: a close differential diagnosis of infantile hypotonia with psychomotor retardation and characteristic facies.

Purine-rich element-binding protein A (PURA) encodes Pur-alpha, a transcriptional activator protein is crucial for normal brain development. Pathogenic variants in PURA are known to cause mental retardation, autosomal dominant 31, characterized by psychomotor delay, absent or poor speech, hypotonia, feeding difficulties, seizures or 'seizure-like' movements, and dysmorphism. PURA-related neurodevelopmental disorder (PURA-related NDD) result either from heterozygous pathogenic sequence variants in PURA or microdeletions spanning PURA. Singleton whole-exome sequencing (WES) was performed for the proband after a clinical diagnosis of infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF) was made. The pathogenic variant was validated by Sanger sequencing in the proband and parents. Comparison of PURA-related NDD and IHPRF was carried out. WES identified a novel, de-novo stop-gain variant c.178G>T in PURA. In addition to typical phenotype, subject also had hypersensitivity to various stimuli which was not reported in PURA-related NDD. Significant phenotypic overlap was observed in subjects with PURA-related NDD and IHPRF especially with IHPRF2, caused by biallelic pathogenic variants in UNC80. This study expands the phenotypic and mutational spectrum of PURA-related NDD. We propose PURA-related NDD to be considered as a close differential diagnosis of IHPRF.

Identification and functional analysis of genetic variants in TBX5 gene promoter in patients with acute myocardial infarction

BackgroundCoronary artery disease (CAD), including acute myocardial infarction (AMI), is a common complex disease. Although a great number of genetic loci and variants for CAD have been identified, genetic causes and underlying mechanisms remain largely unclear. Epidemiological studies have revealed that CAD incidence is strikingly higher in patients with congenital heart disease than that in normal population. T-box transcription factors play critical roles in embryonic development. In particular, TBX5 as a dosage-sensitive regulator is required for cardiac development and function. Thus, dysregulated TBX5 gene expression may be involved in CAD development.MethodsTBX5 gene promoter was genetically and functionally analysed in large groups of AMI patients (n = 432) and ethnic-matched healthy controls (n = 448).ResultsSix novel heterozygous DNA sequence variants (DSVs) in the TBX5 gene promoter (g.4100A > G, g.4194G > A, g.4260 T > C, g.4367C > A, g.4581A > G and g.5004G > T) were found in AMI patients, but in none of controls. These DSVs significantly changed the activity of TBX5 gene promoter in cultured cells (P < 0.05). Furthermore, three of the DSVs (g.4100A > G, g.4260 T > C and g.4581A > G) evidently modified the binding sites of unknown transcription factors.ConclusionsThe DSVs identified in AMI patients may alter TBX5 gene promoter activity and change TBX5 level, contributing to AMI development as a rare risk factor.

Mutant GNLY is linked to Stevens-Johnson syndrome and toxic epidermal necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare severe cutaneous adverse reactions to drugs. Granulysin (GNLY) plays a key role in keratinocyte apoptosis during SJS/TEN pathophysiology. To determine if GNLY-encoding mutations might be related to the protein's functional disturbances, contributing to SJS/TEN pathogenesis, we performed direct sequencing of GNLY's coding region in a group of 19 Colombian SJS/TEN patients. A GNLY genetic screening was implemented in a group of 249 healthy individuals. We identified the c.11G > A heterozygous sequence variant in a TEN case, which creates a premature termination codon (PTC) (p.Trp4Ter). We show that a mutant protein is synthesised, possibly due to a PTC-readthrough mechanism. Functional assays demonstrated that the mutant protein was abnormally located in the nuclear compartment, potentially leading to a toxic effect. Our results argue in favour of GNLY non-synonymous sequence variants contributing to SJS/TEN pathophysiology, thereby constituting a promising, clinically useful molecular biomarker.

Combinatorial interactions of genetic variants in human cardiomyopathy.

Dilated cardiomyopathy (DCM) is a leading cause of morbidity and mortality worldwide; yet how genetic variation and environmental factors impact DCM heritability remains unclear. Here, we report that compound genetic interactions between DNA sequence variants contribute to the complex heritability of DCM. By using genetic data from a large family with a history of DCM, we discovered that heterozygous sequence variants in the TROPOMYOSIN 1 (TPM1) and VINCULIN (VCL) genes cose-gregate in individuals affected by DCM. In vitro studies of patient-derived and isogenic human-pluripotent-stem-cell-derived cardio-myocytes that were genome-edited via CRISPR to create an allelic series of TPM1 and VCL variants revealed that cardiomyocytes with both TPM1 and VCL variants display reduced contractility and sarcomeres that are less organized. Analyses of mice genetically engineered to harbour these human TPM1 and VCL variants show that stress on the heart may also influence the variable penetrance and expressivity of DCM-associated genetic variants in vivo. We conclude that compound genetic variants can interact combinatorially to induce DCM, particularly when influenced by other disease-provoking stressors.

Clinical characteristics and genetic profiles of young and adult patients with cholestatic liver disease

heterozygous ABCB4, ABCB11 and ATP8B1 sequence variants were previously reported to be associated with low phospholipid-associated cholelithiasis, intrahepatic cholestasis of pregnancy, benign recurrent intrahepatic cholestasis and biliary lithiasis. The present study aimed to identify the presence of sequence variations in genes responsible for Mendelian liver disorders in patients with cholestatic liver disease. targeted massive parallel sequencing of a panel of genes involved in bile acid homeostasis was performed in 105 young and adult patients with cholestatic liver disease in our laboratory for molecular diagnosis. The effects of novel variants were evaluated using bioinformatics prediction tools and the Protter and Phyre2 software programs were used to create 2D, 3D topology protein modeling. Genotype-phenotype correlation was established according to molecular analysis and clinical records. twenty novel heterozygous ABCB4 sequence variations, one heterozygous ABCB4 large intragenic deletion and only one novel missense variant in ABCB11 and ATP8B1 were identified. Interestingly, heterozygous and homozygous SLC4A2 missense variants were detected in patients with low phospholipid-associated cholelithiasis. Two patients harbored heterozygous GPBAR1 variants. Common variants such as homozygous ABCB11 p.Val444Ala and heterozygous ABCG8 p.Asp19His were also identified in 12 cases. forty-eight variants were identified in five genes including ABCB4, ABCB11, ATP8B1, SLC4A2 and GPBAR1, twenty-five of which were novel. This study expands the phenotypic and mutational spectrum in genes involved in bile acid homeostasis and highlights the genetic and phenotypic heterogeneity in patients with inherited liver disorders.

Non-ossifying fibroma with a pathologic fracture in a 12-year-old girl with tricho-rhino-phalangeal syndrome: a case report

BackgroundTricho-rhino-phalangeal syndrome (TRPS) is a rare autosomal dominant genetic disorder characterized by distinctive craniofacial and skeletal abnormalities, while non-ossifying fibroma (NOF) is a common benign bone tumour in children and adolescents. To date, no case of TRPS coexisting with NOF has been reported. This report presents a 12-year-old girl who had the characteristic features of tricho-rhino-phalangeal syndrome and non-ossifying fibroma with a fibula fracture.Case presentationA 12-year-old girl was admitted to the Department of Endocrinology and Diabetes for evaluation of brachydactyly and a right fibula fracture. Clinical examination revealed sparse scalp hair, a characteristic bulbous pear-shaped nose, and brachydactyly with significant shortening of the fourth metatarsal. Neither intellectual disability nor multiple exostoses were observed. Radiography of both hands showed brachydactyly and cone-shaped epiphyses of the middle phalanges of the digits of both hands with deviation of the phalangeal axis. Genetic analysis of TRPS1 identified a heterozygous germline sequence variant (p.Ala932Thr) in exon 6 in the girl and her father. Approximately 1 month before being admitted to our department, the girl experienced a minor fall and suffered a fracture of the proximal fibula in the right lower limb. The pathological cytological diagnosis of the osteolytic lesion was NOF. Ten months following the surgery, the lesion on the proximal fibula of the girl disappeared.ConclusionsIn conclusion, the present study is the first to report a rare case of NOF with a pathologic fracture in the fibula of a girl with TRPS. The identification of a missense mutation, (p.Ala932Thr), in exon 6 of TRPS1 in this kindred further suggested that the patient had type I TRPS and indicated that mutations in this exon may be correlated with more pronounced features of the syndrome. Radiological techniques and genetic analysis played key roles in the definitive diagnosis.