Heterozygous Nonsense Variant Research Articles

P-550 Monoallelic CCNB2 variants may cause oocyte maturation defects and embryo developmental arrest

Abstract Study question Have all the genes that may cause embryo development arrest and oocyte maturation defects been identified in female patients? Summary answer We identified a variant in CCNB2 in our patient. To our knowledge, this is first time that meiotic arrest can be caused by mutated-CCNB2. What is known already Successful reproduction involves the maturation of gametes, fertilization, and early development of the embryo. Oocyte maturation involves a sequence of both morphological and molecular transformations, progressing from germinal vesicle oocytes to metaphase I oocytes and ultimately to metaphase II oocytes. Any disruption in the process of oocyte maturation can result in infertility. With the widespread use of whole exome sequencing (WES), several genes that influence oocyte maturation at different stages have been recently identified. Study design, size, duration We here report the case of repeated ART failure due to oocyte maturation defect and embryo developmental arrest. Participants/materials, setting, methods A 30-year-old female and 38-year-old male applied to our clinic due to unexplained infertility for 4 years. The first ART attempt resulted in 2 of 13 oocytes with late MII. During ART performed in our center, germinal vesicle and meiotic arrest were observed in all oocytes. No mature oocytes were obtained. Her grandfather’s sister was also infertile. WES and Sanger analysis were performed to detect any mutation in known/candidate genes and show familial segregation Main results and the role of chance WES analysis revealed no mutation in genes that were previously reported as a cause of oocyte maturation defect or early embryo development in human. We do a further analysis of genes highly or specifically expressed in oocytes or expressed at low levels yet previous studies have suggested its role in embryo development. We identified a c.787C>T, p.Arg263* heterozygous nonsense variant in the CCNB2 gene that was extremely rare in both multiple databases and our in-house database. Multiple lines of computational evidence support the deleterious effect of the variant on the gene product. This variant was transmitted from her healthy father. CCNNB2 is not expressed in any cells other than oocytes in human. The activity of the M phase-promoting factor (MPF) is crucial for advancing through both mitosis and meiosis. CCNB2 plays a vital role by contributing to the activity of pre-MPF during prophase, necessary for generating enough MPF to proceed through maturation. Furthermore, CCNB2 is significantly involved in mouse oocyte meiosis, playing a key role in both the G2/M and MI/anaphase I transitions. Limitations, reasons for caution New disease-causing variants in this gene should be demonstrated in larger cohorts with female infertility. In addition, further functional studies should be carried out both in CCNB2-mutated human and animals. Wider implications of the findings This study may provide a better understanding background of female infertility and the failure of the ART process. CCNB2 may have a crucial role in the oocyte maturation process and can be utilized as a female infertility genetic marker. Trial registration number NA

Variants in HCFC1 and MN1 genes causing intellectual disability in two Pakistani families

BackgroundIntellectual disability (ID) is a neurodevelopmental condition affecting around 2% of children and young adults worldwide, characterized by deficits in intellectual functioning and adaptive behavior. Genetic factors contribute to the development of ID phenotypes, including mutations and structural changes in chromosomes. Pathogenic variants in the HCFC1 gene cause X-linked mental retardation syndrome, also known as Siderius type X-linked mental retardation. The MN1 gene is necessary for palate development, and mutations in this gene result in a genetic condition called CEBALID syndrome.MethodsExome sequencing was used to identify the disease-causing variants in two affected families, A and B, from various regions of Pakistan. Affected individuals in these two families presented ID, developmental delay, and behavioral abnormalities. The validation and co-segregation analysis of the filtered variant was carried out using Sanger sequencing.ResultsIn an X-linked family A, a novel hemizygous missense variant (c.5705G > A; p.Ser1902Asn) in the HCFC1 gene (NM_005334.3) was identified, while in family B exome sequencing revealed a heterozygous nonsense variant (c.3680 G > A; p. Trp1227Ter) in exon-1 of the MN1 gene (NM_032581.4). Sanger sequencing confirmed the segregation of these variants with ID in each family.ConclusionsThe investigation of two Pakistani families revealed pathogenic genetic variants in the HCFC1 and MN1 genes, which cause ID and expand the mutational spectrum of these genes.

Novel insights into the phenotypic spectrum and pathogenesis of Hardikar syndrome

PurposeHardikar syndrome (HS, MIM #301068) is a female-specific multiple congenital anomaly syndrome characterized by retinopathy, orofacial clefting, aortic coarctation, biliary dysgenesis, genitourinary malformations, and intestinal malrotation. We previously showed that heterozygous nonsense and frameshift variants in MED12 cause HS. The phenotypic spectrum of disease and the mechanism by which MED12 variants cause disease is unknown. We aim to expand the phenotypic and molecular landscape of HS and elucidate the mechanism by which MED12 variants cause disease. MethodsWe clinically assembled and molecularly characterized a cohort of 11 previously unreported individuals with HS. Additionally, we studied the effect of MED12 deficiency on ciliary biology, hedgehog, and YAP signaling; pathways implicated in diseases with phenotypic overlap with HS. ResultsWe report novel phenotypes associated with HS, including cardiomyopathy, arrhythmia, and vascular anomalies, and expand the molecular landscape of HS to include splice site variants. We additionally demonstrate that MED12 deficiency causes decreased cell ciliation, and impairs hedgehog and YAP signaling. ConclusionOur data support updating HS standard-of-care to include regular cardiac imaging, arrhythmia screening, and vascular imaging. We further propose that dysregulation of ciliogenesis and YAP and hedgehog signaling contributes to the pathogenesis of HS.

Wolfram Syndrome Type I Case Report and Review-Focus on Early Diagnosis and Genetic Variants.

Background and Objectives: Wolfram syndrome type 1 (OMIM# 222300; ORPHAcode 3463) is an extremely rare autosomal recessive syndrome with a 25% recurrence risk in children. It is characterized by the presence of juvenile-onset diabetes mellitus (DM), progressive optic atrophy (OA), diabetes insipidus (DI), and sensorineural deafness (D), often referred to by the acronym DIDMOAD. It is a severe neurodegenerative disease with a life expectancy of 39 years, with death occurring due to cerebral atrophy. For a positive diagnosis, the presence of diabetes mellitus and optic nerve atrophy is sufficient. The disease occurs because of pathogenic variants in the WFS1 gene. The aim of this article is to present a case report of Wolfram Syndrome Type I, alongside a review of genetic variants, clinical manifestations, diagnosis, therapy, and long-term management. Emphasizing the importance of early diagnosis and a multidisciplinary approach, the study aims to enhance understanding and improve outcomes for patients with this complex syndrome. Materials and Methods: A case of a 28-year-old patient diagnosed with DM at the age of 6 and with progressive optic atrophy at 26 years old is presented. Molecular diagnosis revealed the presence of a heterozygous nonsense variant WFS1 c.1943G>A (p.Trp648*), and a heterozygous missense variant WFS1 c.1675G>C (p.Ala559Pro). Results: The molecular diagnosis of the patient confirmed the presence of a heterozygous nonsense variant and a heterozygous missense variant in the WFS1 gene, correlating with the clinical presentation of Wolfram syndrome type 1. Both allelic variants found in our patient have been previously described in other patients, whilst this combination has not been described before. Conclusions: This case report and review underscores the critical role of early recognition and diagnosis in Wolfram syndrome, facilitated by genetic testing. By identifying pathogenic variants in the WFS1 gene, genetic testing not only confirms diagnosis but also guides clinical management and informs genetic counseling for affected families. Timely intervention based on genetic insights can potentially reduce the progressive multisystem manifestations of the syndrome, thereby improving the quality of life and outcomes for patients.

Novel SPEF2 variants cause male infertility and likely primary ciliary dyskinesia.

This study aimed to identify the genetic causes of male infertility and primary ciliary dyskinesia (PCD)/PCD-like phenotypes in three unrelated Han Chinese families. We conducted whole-exome sequencing of three patients with male infertility and PCD/PCD-like phenotypes from three unrelated Chinese families. Ultrastructural and immunostaining analyses of patient spermatozoa and respiratory cilia and in vitro analyses were performed to analyze the effects of SPEF2 variants. Intracytoplasmic sperm injection (ICSI) was administered to three affected patients. We identified four novel SPEF2 variants, including one novel homozygous splicing site variant [NC_000005.10(NM_024867.4): c.4447 + 1G > A] of the SPEF2 gene in family 1, novel compound heterozygous nonsense variants [NC_000005.10(NM_024867.4): c.1339C > T (p.R447*) and NC_000005.10(NM_024867.4): c.1645G > T (p.E549*)] in family 2, and one novel homozygous missense variant [NC_000005.10(NM_024867.4): c.2524G > A (p.D842N)] in family 3. All the patients presented with male infertility and PCD/likely PCD. All variants were present at very low levels in public databases, predicted to be deleterious in silico prediction tools, and were further confirmed deleterious by in vitro analyses. Ultrastructural analyses of the spermatozoa of the patients revealed the absence of the central pair complex in the sperm flagella. Immunostaining of the spermatozoa and respiratory cilia of the patients validated the pathogenicity of the SPEF2 variants. All patients carrying SPEF2 variants underwent one ICSI cycle and delivered healthy infants. Our study reported four novel pathogenic variants of SPEF2 in three male patients with infertility and PCD/PCD-like phenotypes, which not only extend the spectrum of SPEF2 mutations but also provide information for genetic counseling and treatment of such conditions.

A novel variant of DNM1L expanding the clinical phenotypic spectrum: a case report and literature review

BackgroundMitochondrial diseases are heterogeneous in terms of clinical manifestations and genetic characteristics. The dynamin 1-like gene (DNM1L) encodes dynamin-related protein 1 (DRP1), a member of the GTPases dynamin superfamily responsible for mitochondrial and peroxisomal fission. DNM1L variants can lead to mitochondrial fission dysfunction.Case presentationHerein, we report a distinctive clinical phenotype associated with a novel variant of DNM1L and review the relevant literature. A 5-year-old girl presented with paroxysmal hemiplegia, astigmatism, and strabismus. Levocarnitine and coenzyme Q10 supplement showed good efficacy. Based on the patient’s clinical data, trio whole-exome sequencing (trio-WES) and mtDNA sequencing were performed to identify the potential causative genes, and Sanger sequencing was used to validate the specific variation in the proband and her family members. The results showed a novel de novo heterozygous nonsense variant in exon 20 of the DNM1L gene, c.2161C>T, p.Gln721Ter, which is predicted to be a pathogenic variant according to the ACMG guidelines. The proband has a previously undescribed clinical manifestation, namely hemiparesis, which may be an additional feature of the growing phenotypic spectrum of DNM1L-related diseases.ConclusionOur findings elucidate a novel variant in DNM1L-related disease and reveal an expanding phenotypic spectrum associated with DNM1L variants. This report highlights the necessity of next generation sequencing for early diagnosis of patients, and that further clinical phenotypic and genotypic analysis may help to improve the understanding of DNM1L-related diseases.

Identification of novel SHANK2 variants in two Chinese families via exome and RNA sequencing

BackgroundSHANK2 encodes a postsynaptic scaffolding protein involved in synapse formation, stabilization and homeostasis. Variations or microdeletions in the SHANK2 gene have been linked to a variety of neurodevelopmental disorders, including autism spectrum disorders (ASD) and mild to moderate intellectual disability (ID) in human. However, the number of reported cases with SHANK2 defects remains limited, with only 14 unrelated patients documented worldwide.MethodsIn this study, we investigated four patients from three families with ID. Whole-exome sequencing (WES) was performed to explore the genetic causes, while Sanger sequencing was used to confirm the identified variants. Furthermore, RNA sequencing and functional enrichment analysis were performed on patients with likely pathogenic variants to gain further insights into the molecular landscape associated with these variants.ResultsTwo novel variants in the SHANK2 gene: a heterozygous splicing substitution (NM_012309.5:c.2198-1G>A p.Pro734Glyfs*22) in Family 1, and a heterozygous nonsense variant [NM_012309.5:c.2310dupT p.(Lys771*)] in Family 2 were identified by WES and confirmed by Sanger sequencing. RNA sequencing and cohort analysis identified a total of 1,196 genes exhibiting aberrant expression in three patients. Functional enrichment analysis revealed the involvement of these genes in protein binding and synaptic functions.ConclusionWe identified two novel loss of function variants that broadens the spectrum of SHANK2 variants. Furthermore, this study enhances our understanding of the molecular mechanisms underlying SHANK2-related disorders.

FRI692 Severe Hyperparathyroidism And Nephrolithiasis In An Adolescent Male Due To CDC73 Positive Parathyroid Carcinoma

Abstract Disclosure: J.T. Nguyen: None. D.H. Geller: None. A. Ryabets-Lienhard: None. Introduction: Parathyroid carcinoma is a rare cause of primary hyperparathyroidism in the pediatric population, with only 20 previously reported cases. Variations in the CDC73 gene have been associated with sporadic parathyroid carcinoma, autosomal dominant hyperparathyroidism-jaw tumor syndrome (HPT-JT), and familial isolated hyperparathyroidism (FIHP). To our knowledge, only 3 cases of pediatric parathyroid carcinoma with confirmed variants of CDC73 have been reported. We present a case of severe hyperparathyroidism and nephrolithiasis due to parathyroid carcinoma caused by a pathogenic variant in CDC73 in a pediatric patient, highlighting the importance of early recognition and management. Clinical Case: An otherwise asymptomatic 14-year-old male presented with one year of recurrent nephrolithiasis. Initial tests demonstrated hypercalcemia (13.8 mg/dL, 8.4-10.2 mg/dL), normal phosphorous, elevated PTH (1,379 pg/mL, 7.5-53.5 pg/mL), and elevated alkaline phosphatase (514 U/L, 30-250 U/L). The hypercalcemia was treated with hyperhydration, furosemide, and pamidronate. Sestamibi scan revealed a focus of abnormal uptake suggestive of a left parathyroid mass, with ultrasonographic demonstration of a predominantly solid, hypoechoic, and vascular mass centered posterior to the inferior left thyroid lobe. Skeletal survey was notable for trabecular abnormalities and osteopenia. He underwent a left parathyroid mass excision. Final pathology confirmed the parathyroid carcinoma, with multiple foci of vascular invasion without evidence of capsular involvement. Immunohistochemistry demonstrated diffuse positivity for PTH and loss of parafibromin expression by the tumor cells. Genetic testing revealed a pathogenic germline heterozygous nonsense variant in CDC73 (c.376C>T(p.Arg126Ter)) resulting in protein truncation. Post-operative PTH was undetectable with normal calcium and phosphorous. Calcitriol, cholecalciferol, and calcium carbonate supplementation were implemented to prevent further hypocalcemia due to transient hypoparathyroidism and hungry bone syndrome. Calcium supplementation was discontinued within one year of his surgery. Serial thyroid ultrasounds and CT scans have been negative for recurrent or residual disease without evidence of metastasis. Mandibular x-ray showed no evidence of tumors or lesions involving the jaw. He has been noted to have small right renal cysts without other kidney abnormalities. Conclusion: Severe hyperparathyroidism due to parathyroid carcinoma can present as recurrent nephrolithiasis in an otherwise asymptomatic individual. Pediatric patients with a longstanding history of kidney stones should undergo screening for hypercalcemia and hyperparathyroidism even in the absence of other signs and symptoms of elevated calcium to ensure timely diagnosis and appropriate management. Presentation: Friday, June 16, 2023

Identification of the first homozygous intragenic deletion in the YY1AP1 gene in a consanguineous family: New insights into the phenotypic variability associated with Grange syndrome.

Grange syndrome (GRNG-MIM#135580) is a rare recessive disorder associating variable features including diffuse vascular stenosis, brachysyndactyly, osteopenia with increased bone fragility, cardiac malformations, and variable developmental delay. Since its first description in 1998, only 15 individuals from 10 families have been reported, carrying homozygous or compound heterozygous frameshift or nonsense variants in YY1AP1. In a patient with cutaneous and bone syndactyly and a hemorrhagic stroke at the age of 16 months, consistent with a clinical diagnosis of GRNG, we performed exome sequencing after negative array-CGH and congenital limb malformation panel results. Copy number variant analysis from exome data identified a homozygous intragenic out-of-frame deletion of 1.84 kb encompassing exons seven and eight of YY1AP1, confirming a molecular diagnosis of GRNG. Genetic counseling led to the identification of additional family members compatible with GRNG. Here, we provide new insights into the phenotypic variability associated with GRNG and highlight the utility of the detection of small copy number variants to identify the molecular causes of heterogeneous malformative genetic disorders.

Retinopathy as an initial sign of hereditary immunological diseases: report of six families and challenges in eye clinic.

Retinal degenerative or inflammatory changes may occur with hereditary immunological disorders (HID) due to variants in approximately 20 genes. This study aimed to investigate if such retinopathy may present as an initial sign of immunological disorders in eye clinic. The variants in the 20 genes were selected from in-house exome sequencing data from 10,530 individuals with different eye conditions. Potential pathogenic variants were assessed by multistep bioinformatic analysis. Pathogenic variants were defined according to the ACMG/AMP criteria and confirmed by Sanger sequencing, co-segregation analysis, and consistency with related phenotypes. Ocular clinical data were thoroughly reviewed, especially fundus changes. A total of seven pathogenic variants in four of the 20 genes were detected in six probands from six families, including three with hemizygous nonsense variants p.(Q308*), p.(Q416*), and p.(R550*) in MSN, one with homozygous nonsense variants p.(R257*) in AIRE, one with compound heterozygous nonsense variants p.(R176*) and p.(T902*) in LAMB2, and one with a known c.1222T>C (p.W408R) heterozygous variant in CBL. Ocular presentation, as the initial signs of the diseases, was mainly retinopathy mimicking other forms of hereditary retinal degeneration, including exudative vitreoretinopathy in the three patients with MSN variants or tapetoretinal degeneration in the other three patients. Neither extraocular symptoms nor extraocular manifestations were recorded at the time of visit to our eye clinic. However, of the 19 families in the literature with retinopathy caused by variants in these four genes, only one family with an AIRE homozygous variant had retinopathy as an initial symptom, while the other 18 families had systemic abnormalities that preceded retinopathy. This study, for the first time, identified six unrelated patients with retinopathy as their initial and only presenting sign of HID, contrary to the previous reports where retinopathy was the accompanying sign of systemic HID. Recognizing such phenotype of HID may facilitate the clinical care of these patients. Follow-up visits to such patients and additional studies are expected to validate and confirm our findings.

Clinical and molecular characterization of parathyroid carcinoma in multiple endocrine neoplasia type 1.

Nineteen cases of parathyroid carcinoma in patients with multiple endocrine neoplasia type 1 have been reported in the literature, of which 11 carry an inactivating germline mutation in the MEN1 gene. Somatic genetic abnormalities in these parathyroid carcinomas have never been detected. In this paper, we aimed to describe the clinical and molecular characterization of a parathyroid carcinoma identified in a patient with MEN1. A 60-year-old man was diagnosed with primary hyperparathyroidism during the postoperative period of lung carcinoid surgery. Serum calcium and parathyroid hormone levels were 15.0 mg/dL (8.4-10.2) and 472 pg/mL (12-65), respectively. The patient underwent parathyroid surgery, and histological findings were consistent with parathyroid carcinoma. Analysis of the MEN1 gene by next-generation sequencing (NGS) identified a novel germline heterozygous nonsense pathogenic variant (c.978C>A; p.(Tyr326*)), predicted to encode a truncated protein. Genetic analysis of the parathyroid carcinoma revealed a c.307del, p.(Leu103Cysfs*16) frameshift truncating somatic MEN1 variant in the MEN1 gene, which is consistent with MEN1 tumor-suppressor role, confirming its involvement in parathyroid carcinoma etiology. Genetic analysis of CDC73, GCM2, TP53, RB1, AKT1, MTOR, PIK3CA and CCND1 genes in the parathyroid carcinoma DNA did not detect any somatic mutations. To our knowledge, this is the first report of a PC case presenting both germline (first-hit) and somatic (second-hit) inactivation of the MEN1 gene.

Heterozygous nonsense variants in laminin subunit 3α resulting in Ebstein’s anomaly

Ebstein's anomaly is a rare congenital heart disease characterized by tricuspid valve downward displacement and is associated with additional cardiac phenotypes such as left ventricle non-compaction. The genetic basis of Ebstein's anomaly has yet to be fully elucidated, although several genes (e.g., NKX2-5, MYH7, TPM1, and FLNA) may contribute to Ebstein's anomaly. Here, in two Ebstein's anomaly families (a three-generation family and a trio), we identified independent heterozygous nonsense variants in laminin subunit 3 (LAMA3), cosegregated with phenotypes in families with reduced penetrance. Furthermore, knocking out Lama3 in mice revealed that haploinsufficiency of Lama3 led to Ebstein's malformation of the tricuspid valve and an abnormal basement membrane structure. In conclusion, we identified a novel gene-disease association of LAMA3 implicated in Ebstein's anomaly, and the findings extended our understanding of the role of the extracellular matrix in Ebstein's anomaly etiology.

A Novel Variant in the PAX4 Gene Causes Maturity-Onset Diabetes of the Young (MODY), Type IX with Neurodevelopmental Disorder

A genetically diverse condition, maturity-onset diabetes of the young (MODY), frequently develops before the age of 25. MODY is caused by disease-causing sequence variations in the PAX4 gene, which is found on chromosome 7q32.1. Additionally, it has also been observed that variants in PAX4 have also been associated with neurodevelopmental disability. Whole exome sequencing (WES) followed by Sanger sequencing was performed for all the available affected and unaffected members of the family. Data analysis revealed a novel heterozygous nonsense variant (c.61C>T; p.Gln21*) in the PAX4 gene in the affected individuals, which segregated perfectly with the disease phenotype. The present study adds to the PAX4 mutation spectrum and reports on the first case of MODY associated with neurodevelopmental disorders in humans.

Restoration of functional PAX6 in aniridia patient iPSC-derived ocular tissue models using repurposed nonsense suppression drugs

Congenital aniridia is a rare, pan-ocular disease causing severe sight loss, with only symptomatic intervention offered to patients. Approximately 40% of aniridia patients present with heterozygous nonsense variants in PAX6, resulting in haploinsufficiency. Translational readthrough-inducing drugs (TRIDs) have the ability to weaken the recognition of in-frame premature termination codons (PTCs), permitting full-length protein to be translated. We established induced pluripotent stem cell (iPSC)-derived 3D optic cups and 2D limbal epithelial stem cell (LESC) models from two aniridia patients with prevalent PAX6 nonsense mutations. Both invitro models show reduced PAX6 protein levels, mimicking the disease. The repurposed TRIDs amlexanox and 2,6-diaminopurine (DAP) and the positive control compounds ataluren and G418 were tested for their efficiency. Amlexanox was identified as the most promising TRID, increasing full-length PAX6 levels in both models and rescuing the disease phenotype through normalization of VSX2 and cell proliferation in the optic cups and reduction of ABCG2 protein and SOX10 expression in LESCs. This study highlights the significance of patient iPSC-derived cells as a new model system for aniridia and proposes amlexanox as a new putative treatment for nonsense-mediated aniridia.

#3460 DEVELOPMENT OF A NEW CELLULAR MODEL TO EVALUATE THE CLINICAL IMPACT OF PKD1 VARIANTS EXPLOITING CRISPR/CAS9 SYSTEM

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common single-gene disorder and the most frequent progressive kidney disease, which ultimately leads to kidney failure and renal replacement therapy. Almost 80% of cases of ADPKD are attributed to germline mutations in PKD1, even though at least one second somatic event such as the inactivation of the remaining wild-type PKD1 allele is required for the disease's manifestation. A significant portion of all variants identified in PKD1 is classified as “variant of unknown significance” (VUS) and understanding their functional impact may be of diagnostic importance for patients and their families. So, a proper experimental model to study the functional impact of different genetic lesions is needed to readily confirm their pathogenicity. Method The HEK293T cell line was modified to express an inducible Cas9 and afterwards transfected with a sgRNA targeting exon 15 of PKD1 to generate clones carrying homozygous or heterozygous nonsense variants, validated by Sanger sequencing. Transcript level and protein expression were evaluated, and then functional read-outs were set-up to validate the model. Heterozygous clones were used to introduce a second PKD1 variant, c.11614G>A p.(Glu3872Lys) in exon 42, using a modified CRISPR system called base editors, able to operate single-base substitutions (Figure 1A). Results Heterozygous (+/−) and homozygous (−/−) exon 15 PKD1 clones were generated. These cells showed a slight decrement in PKD1 mRNA levels, and PC1 protein loss was confirmed in full knock-out clones. Functionally, immunofluorescence staining highlighted a different cytoskeletal rearrangement in cells lacking PC1, which did not present actin protrusions at variance with WT and heterozygous cells. In agreement with these results, PKD1−/− clones showed reduced migration as demonstrated by wound healing assay. In starving conditions, PKD1−/− cells were characterized by increased viability, resistance to cell death and a disrupted autophagic pathway, as demonstrated by LC3BI-II conversion, which in PKD1−/− cells is diminished. Validation of the model is also supported by RNA sequencing data indicating that double knock-out cell lines display an upregulation of genes involved in proliferative pathways and epithelial-mesenchymal transition, as well as a down-regulation of genes involved in cytoskeletal organization. We then used the heterozygous exon 15 clones to introduce a specific variant in exon 42, classified as C4 according to ACMG criteria. To do so, PKD1+/+ and PKD1+/− cell lines were transfected with a sgRNA guide that correctly positioned the Cytosine Base Editor. Using this approach we could generate a double knock-out cell line on exon 42 and a “double hit” cell line carrying a heterozygous variant on exon 15 and an homozygous one on exon 42, which represents a more likely real-life situation. Functional assessment of the variant's pathogenicity and comparison with exon 15 double knock-outs is currently ongoing. Conclusion In conclusion, we generated a new PKD cellular model that can be easily exploited to reproduce some of the VUS variants identified, by clinical exome sequencing, in our cohort of ADPKD patients. Results obtained provide a proof-of-principle of the feasibility of this approach and allowed to identify selective read-outs (Figure 1B) to be used for a rapid screening to assess the phenotypic impact of specific PKD1 variants.

PKD1 Nonsense Variant in a Lagotto Romagnolo Family with Polycystic Kidney Disease.

A female Lagotto Romagnolo dog with polycystic kidney disease (PKD) and her progeny, including PKD-affected offspring, were studied. All affected dogs appeared clinically inconspicuous, while sonography revealed the presence of renal cysts. The PKD-affected index female was used for breeding and produced two litters with six affected offspring of both sexes and seven unaffected offspring. The pedigrees suggested an autosomal dominant mode of inheritance of the trait. A trio whole genome sequencing analysis of the index female and her unaffected parents identified a de novo heterozygous nonsense variant in the coding region of the PKD1 gene. This variant, NM_001006650.1:c.7195G>T, is predicted to truncate 44% of the open reading frame of the wild-type PKD1 protein, NP_001006651.1:p.(Glu2399*). The finding of a de novo variant in an excellent functional candidate gene strongly suggests that the PKD1 nonsense variant caused the observed phenotype in the affected dogs. Perfect co-segregation of the mutant allele with the PKD phenotype in two litters supports the hypothesized causality. To the best of our knowledge, this is the second description of a PKD1-related canine form of autosomal dominant PKD that may serve as an animal model for similar hepatorenal fibrocystic disorders in humans.

Case report: Compound heterozygous nonsense PCDH15 variant and a novel deep-intronic variant in a Chinese child with profound hearing loss.

Usher syndrome is a condition characterized by partial or total hearing loss and progressive pigmentary retinopathy. Usher syndrome type 1F is caused by biallelic loss-of-function variants in Protocadherin 15 (PCDH15), which encodes the PCDH15 protein that plays an important role in the morphogenesis and cohesion of stereocilium bundles and retinal photoreceptor cell maintenance and function. We report a child with bilateral nonsyndromic sensorineural hearing loss who received an inconclusive diagnosis based on clinical gene panel testing, which identified a paternal heterozygous nonsense variant (NM_033056.4: c.733C>T, p.R245*) in PCDH15. This variant has been described as a founder variant in the Ashkenazi Jewish population. A novel deep-intronic variant (NM_033056.4: c.705+3767_705+3768del) inherited from the patient's mother was identified by trio-based whole-genome sequencing (WGS). A minigene splicing assay revealed that c.705+3767_705+3768del results in aberrant retention of 50 or 68 bp of intron 7. Our genetic test results provided precise genetic counseling and prenatal diagnosis for this family, and our findings highlight the power of WGS for detecting deep-intronic variants in patients with undiagnosed rare diseases. Additionally, this case expands the variant spectrum of the PCDH15 gene and our results support the extremely low carrier frequency of c.733C>T in the Chinese population.

A Heterozygous Nonsense Variant of NFKBIA Leading to Gain-of-Function

The NFKBIA gene encodes the protein IκBα, an important regulatory protein that interacts with nuclear factor kappa B family of transcription factors required for innate and adaptive immune responses and normal ectodermal development. Heterozygous nonsense and missense variants of the NFKBIA gene have been reported in a total of 19 individuals with autosomal dominant ectodermal dysplasia with immunodeficiency (EDA-ID) since 2003. Here, we report a patient with a novel de novo heterozygous nonsense variant in the N-terminal of NFKBIA. This patient initially presented to Immunology due to an abnormal presentation of IgA nephropathy (IgAN) with solely kappa staining and a notable history of recurrent AOM, PNA and viral infections including septic shock, diffuse watershed ischemic injury and new seizure onset due to influenza A. Immunologic assessment revealed lymphocytosis of T- and B-cells, particularly CD4+ and CD8+ memory, and CD27-IgD+ cells. In contrast, class-switched, non-class-switched memory B-cells, CD21lo, and plasmablasts were low. A distinct B-cell population expressing less CD19 with negative surface IgD, IgG, and IgM was also observed. Roughly 50% of these cells had IgM expression without surface IgD and were CD24 and CD27 negative, CD21 lo, and CD28 mid. Functionally, T-cells had poor response and proliferation to PHA as well as soluble anti-CD3, anti-CD3+anti-CD28, and anti-CD3+IL-2. Vaccine response was diminished, particularly to polysaccharides. Furthermore, TLR function of PBMCs were assessed revealing decreased cytokine response to soluble ligand exposure. Physical exam findings consistent with EDA-ID included several absent and conical teeth, xerosis, and sparse hair with a history notable for heat intolerance. In line with prior reports of patients with early truncating variants in NFKBIA, the infectious manifestations in these patients have been mild, particularly in comparison to those with missense variants that affect serine 32. In this patient, HSCT was not undertaken, but rather IgG replacement therapy was initiated resulting in absence of any further serious infections. Additionally, glucocorticoid therapy was initiated for IgAN with subsequent sustained remission. With an increase in published data, this may help broaden our scope of what phenotypical presentation can be expected, which may help increase the rate of diagnosis and early identification.

PO-04-015 BIVENTRICULAR ARRHYTHMOGENIC CARDIOMYOPATHY ASSOCIATED WITH A NOVEL HETEROZYGOUS PLAKOPHILIN-2 EARLY TRUNCATING VARIANT

N/A N/A N/A A 31-year-old male patient suffered from palpitations, which occurred particularly during and shortly after physical activity. The patient was very athletic and took part in marathons. His family history was unremarkable. The electrocardiogram showed T wave inversions in V1-V3 (Figure 1A). During bicycle ergometry monomorphic premature ventricular complexes (PVC) and non-sustained ventricular tachycardia episodes (left-bundle branch block morphology, inferior axis) evolved (Figure 1B, black arrow). Of note, under maximal physical stress a second PVC with a right-bundle branch block morphology occurred (Figure 1B, red arrow) suggesting left ventricular (LV) involvement. Echocardiographic evaluation showed a dilated right ventricle (RV) with reduced systolic function, a subtricuspid aneurysm and multiple sacculations of the RV free wall (Figure 1C-D, white markers). Cardiac magnetic resonance imaging (MRI) confirmed the right ventricular findings (Figure 1E, red arrows). In addition, late gadolinium enhancement and fatty deposits were visible in the LV lateral wall (Figure 1F-G, yellow arrows and blue asterix). Genetic testing was performed using a panel covering 173 genes (Agilent, SureSelectQXTTarget Enrichment) and next generation sequencing (NGS) (Illumina MiSeq). It revealed a novel heterozygous nonsense variant in the plakophilin-2 (PKP2) gene (808C>T p. (Gln270Ter) in exon 3) that causes an early truncation, which was confirmed by Sanger sequencing. It was classified as pathogenic (class V). Cascade screening of the asymptomatic patient’s mother (64-y-old), father (70-y-old) and sister (33-y-old) showed that the patient's father and sister harbored the same PKP-2 variant. Both did not report a history of participating in competitive sports, and thorough cardiac evaluation of these two genotype positive family members showed normal findings. Previous studies have indicated that early truncation of the PKP2 C-terminus likely causes arrhythmogenic right ventricular cardiomyopathy (ARVC) irrespective of transcript position. Although desmosomal and non-desmosomal variants create certain genetic potential for the development of ARVC, exercise has an important role in determining the development, severity, and pattern of phenotypic expression. The fact that the only index patient, as a marathon runner, developed ARVC, suggests that the novel PKP2 variant poses a genetic risk, but is not sufficient for inducing an ARVC phenotype by itself.

Familial hypotrichosis simplex of the scalp associated with a novel heterozygous nonsense variant in CDSN.

This report describes a case of an 18-year-old white British woman with HTSS1, whose phenotype was characterized by the inability to grow long scalp hair. Whole exome sequencing identified a novel pathogenic heterozygous nonsense variant (NM_001264.4: c.484C>T, NP_001255.3: p.Gln162Ter) in CDSN, which encodes corneodesmosin. HTSS1, described in this patient’s case, showed distinct clinical and histopathological features, thereby expanding the genotype–phenotype paradigm of HTSS1.