Gene Panel Analysis Research Articles

Successful eculizumab treatment as an adjunctive therapy to desensitization in ABO-incompatible living donor kidney transplantation and its molecular phenotypes

IntroductionABO-incompatible (ABOi) kidney transplantation (KT) has become an important option to overcome organ shortage. Plasmapheresis/rituximab-based desensitization therapy has successfully reduced anti-ABO antibody levels and suppressed antibody-mediated rejection (AMR) in ABOi KT. However, high titers of anti-ABO antibodies in some patients are refractory to standard desensitization, leading to loss of KT opportunities or AMR.MethodsEculizumab treatment was used an adjunctive therapy to rescue high-titer ABOi KT patients refractory to plasmapheresis/rituximab-based desensitization. Molecular phenotypes of allograft biopsies and cellular phenotypes of peripheral blood mononuclear cells of eculizumab group were compared with those of control groups using the Banff Human Organ Transplant gene panel and flow-cytometric analysis, respectively.ResultsThe initial titers of anti-ABO antibodies in the two patients were 1:512 and >1:1024; the final pre-transplant titers after desensitization were 1:128 and 1:64. Both patients received eculizumab from KT day to two or four weeks post-KT and maintained stable renal function up to one-year post-transplantation without overt infection, despite early episodes of probable AMR or borderline T cell-mediated rejection. Molecular phenotype analysis revealed that gene expression patterns in the ABOi KT with eculizumab group overlapped with those in the ABOi KT with AMR group more than in the ABOi KT without AMR group, except for complement pathway-related gene expression. Anti-ABO antibody titers decreased to low levels 1–3 months post-transplant in the eculizumab group in parallel with decreasing anti-B-specific B cells.ConclusionsShort-term eculizumab therapy is promising for rescuing ABOi KT recipients with high anti-ABO antibody titers refractory to plasmapheresis-based desensitization therapy.

Exome variant prioritization in a large cohort of hearing-impaired individuals indicates IKZF2 to be associated with non-syndromic hearing loss and guides future research of unsolved cases.

Although more than 140 genes have been associated with non-syndromic hereditary hearing loss (HL), at least half of the cases remain unexplained in medical genetic testing. One reason is that pathogenic variants are located in 'novel' deafness genes. A variant prioritization approach was used to identify novel (candidate) genes for HL. Exome-wide sequencing data were assessed for subjects with presumed hereditary HL that remained unexplained in medical genetic testing by gene-panel analysis. Cases in group AD had presumed autosomal dominantly inherited HL (n = 124), and in group AR, presumed autosomal recessive HL (n = 337). Variants in known and candidate deafness genes were prioritized based on allele frequencies and predicted effects. Selected variants were tested for their co-segregation with HL. Two cases were solved by variants in recently identified deafness genes (ABHD12, TRRAP). Variant prioritization also revealed potentially causative variants in candidate genes associated with recessive and X-linked HL. Importantly, missense variants in IKZF2 were found to co-segregate with dominantly inherited non-syndromic HL in three families. These variants specifically affected Zn2+-coordinating cysteine or histidine residues of the zinc finger motifs 2 and 3 of the encoded protein Helios. This finding indicates a complex genotype-phenotype correlation for IKZF2 defects, as this gene was previously associated with non-syndromic dysfunction of the immune system and ICHAD syndrome, including HL. The designed strategy for variant prioritization revealed that IKZF2 variants can underlie non-syndromic HL. The large number of candidate genes for HL and variants therein stress the importance of inclusion of family members for variant prioritization.

Advancing Precision Oncology: Whole-Exome Sequencing in Endometrial Cancer Liquid-Based Cytology.

Diagnostic strategies for endometrial cancer have been evolving, with cytologic analysis being considered a key method in integrated oncologic diagnostics because of its less invasive nature and adaptability to various assessments. Liquid-based cytology (LBC) has emerged as a promising method for intact DNA preservation; it exhibits improved efficiency in advanced sequencing applications such as next-generation sequencing. However, despite the use of LBC in panel assays, its application in whole-exome sequencing (WES) for comprehensive genomic profiling remains underexplored. To investigate whether molecular classification is possible based on WES using DNA derived from LBC specimens. We combined WES with targeted gene panel analysis to compare genomic findings of LBC and traditional tissue samples obtained from 7 cases of endometrial cancer. We investigated pathogenic mutations, tumor mutational burden, and microsatellite instability, and achieved molecular classification with high accuracy. We found a substantial concordance between LBC and traditional tissue samples in terms of pathogenic mutation detection, with a 95% match in the LBC samples and 94% in the tissue samples. Notably, our results highlight the importance of combining WES with panel-based analysis in identifying the ultramutated status of a case that had been missed during panel analysis. Our findings emphasize the potential of LBC samples in the precise and noninvasive genomic analysis of cases of endometrial cancer and offer a new avenue for developing diagnostic and therapeutic strategies in precision oncology.

9293 Do Obesity-Associated MRAP2 Variants Modulate MC4R and GHSR Signaling?

Abstract Disclosure: A.V. Rodriguez Rondon: None. F. Volker: None. M.S. Welling: None. C. de Groot: None. M.M. van Haelst: None. E.L. van den Akker: None. E.F. van Rossum: None. P.J. Delhanty: None. J.A. Visser: None. Melanocortin 2 receptor accessory protein 2 (MRAP2) is a membrane-bound protein expressed on hypothalamic neurons that modulate the function of appetite-regulating receptors, including melanocortin-4 receptor (MC4R) and growth hormone secretagogue receptor (GHSR). While pathogenic variants in the MRAP2 gene are associated with obesity, the mechanism for this remains unclear. The aim of this study is to investigate the functional impact of wildtype (WT) MRAP2 and MRAP2 variants on MC4R and GHSR signaling. Four MRAP2 variants (S80F, R125C, P167A and Q174X) were identified in patients with obesity by obesity gene panel analysis. The functional effects of these variants were analyzed by co-transfecting HEK293 cells with expression plasmids encoding WT or variant MRAP2 and WT MC4R or WT GHSR. Endpoint analyses included cell surface expression, and ligand-induced second messenger responses (α-MSH-induced cAMP response for MC4R and ghrelin-induced Ca2+ mobilization for GHSR), β-arrestin-2 recruitment, and internalization. WT MRAP2 significantly decreased basal MC4R cell surface expression by 68.1±4.3% (p<0.001). In the absence of MRAP2, α-MSH induced MC4R internalization. However, in the presence of WT MRAP2, MC4R cell surface expression was increased by 89.4±6.9% upon α-MSH stimulation (p<0.001). GHSR cell surface expression and ghrelin-induced internalization were not affected by WT MRAP2. Furthermore, in the cells expressing MC4R with WT MRAP2, the maximal α-MSH-induced cAMP and β-arrestin-2 recruitment responses were increased by 29.8±5.2% and 81.3±4.2% respectively (p<0.001), compared with those lacking MRAP2. We also investigated GHSR ghrelin-induced Ca2+ mobilization and found that, in the presence of WT MRAP2, this increased significantly by 95.37±21.9%, (p<0.0003). However, ghrelin-induced β-arrestin-2 recruitment was suppressed by 69.8±7.6%, (p<0.001). Using these endpoints we found no effect of the variants on the ability of MRAP2 to modulate ligand-induced signaling by MC4R and GHSR. Our results indicate that WT MRAP2 enhances the cAMP response by increasing MC4R cell membrane expression upon α-MSH stimulation, and enhances GHSR Ca2+ mobilization by decreasing β-arrestin-2 recruitment upon ghrelin stimulation. The MRAP2 variants assessed in this study behaved as WT MRAP2 in α-MSH-induced MC4R and ghrelin-induced GHSR signaling, suggesting that these may be benign variants. However, since MRAP2 can modulate multiple receptors, we cannot rule out that these MRAP2 variants affect other appetite-regulating receptors and thereby influence body weight regulation via other pathways. Presentation: 6/3/2024

6789 Do Obesity-Associated MRAP2 Variants Modulate MC4R and GHSR Signaling?

Abstract Disclosure: A.V. Rodriguez Rondon: None. F. Volker: None. M.S. Welling: None. C. de Groot: None. M.M. van Haelst: None. E.L. van den Akker: None. E.F. van Rossum: None. P.J. Delhanty: None. J.A. Visser: None. Melanocortin 2 receptor accessory protein 2 (MRAP2) is a membrane-bound protein expressed on hypothalamic neurons that modulate the function of appetite-regulating receptors, including melanocortin-4 receptor (MC4R) and growth hormone secretagogue receptor (GHSR). While pathogenic variants in the MRAP2 gene are associated with obesity, the mechanism for this remains unclear. The aim of this study is to investigate the functional impact of wildtype (WT) MRAP2 and MRAP2 variants on MC4R and GHSR signaling. Four MRAP2 variants (S80F, R125C, P167A and Q174X) were identified in patients with obesity by obesity gene panel analysis. The functional effects of these variants were analyzed by co-transfecting HEK293 cells with expression plasmids encoding WT or variant MRAP2 and WT MC4R or WT GHSR. Endpoint analyses included cell surface expression, and ligand-induced second messenger responses (α-MSH-induced cAMP response for MC4R and ghrelin-induced Ca2+ mobilization for GHSR), β-arrestin-2 recruitment, and internalization. WT MRAP2 significantly decreased basal MC4R cell surface expression by 68.1±4.3% (p<0.001). In the absence of MRAP2, α-MSH induced MC4R internalization. However, in the presence of WT MRAP2, MC4R cell surface expression was increased by 89.4±6.9% upon α-MSH stimulation (p<0.001). GHSR cell surface expression and ghrelin-induced internalization were not affected by WT MRAP2. Furthermore, in the cells expressing MC4R with WT MRAP2, the maximal α-MSH-induced cAMP and β-arrestin-2 recruitment responses were increased by 29.8±5.2% and 81.3±4.2% respectively (p<0.001), compared with those lacking MRAP2. We also investigated GHSR ghrelin-induced Ca2+ mobilization and found that, in the presence of WT MRAP2, this increased significantly by 95.37±21.9%, (p<0.0003). However, ghrelin-induced β-arrestin-2 recruitment was suppressed by 69.8±7.6%, (p<0.001). Using these endpoints we found no effect of the variants on the ability of MRAP2 to modulate ligand-induced signaling by MC4R and GHSR. Our results indicate that WT MRAP2 enhances the cAMP response by increasing MC4R cell membrane expression upon α-MSH stimulation, and enhances GHSR Ca2+ mobilization by decreasing β-arrestin-2 recruitment upon ghrelin stimulation. The MRAP2 variants assessed in this study behaved as WT MRAP2 in α-MSH-induced MC4R and ghrelin-induced GHSR signaling, suggesting that these may be benign variants. However, since MRAP2 can modulate multiple receptors, we cannot rule out that these MRAP2 variants affect other appetite-regulating receptors and thereby influence body weight regulation via other pathways. Presentation: 6/3/2024

MCM9 compound heterozygosity in an adolescent with premature ovarian insufficiency.

Delayed puberty in girls is often related to late maturation but is occasionally the first sign of premature ovarian insufficiency (POI). POI is a condition that affects ovarian function and fertility, and its etiology is unknown in most cases. Genetic factors have recently been identified in 20-25% of women with POI, involving genes that regulate various aspects of ovarian development and maintenance. We report a case of delayed puberty due to POI in an adolescent from a non-consanguineous family who carried two variants in the MCM9 gene. MCM9 is essential for DNA replication and repair, and its dysfunction can lead to chromosomal instability and ovarian failure. Our case highlights the importance of targeted gene panel analysis, particularly in POI patients with negative autoimmunity screening, and evidence of ovarian or uterine dysgenesis on pelvic imaging. Delayed puberty in girls is often self-limiting, but it can also indicate underlying conditions with lifelong implications, such as premature ovarian insufficiency (POI). Patients with POI, negative autoimmune screening, a normal karyotype, and no FMR premutation should undergo further genetic testing, preferably through targeted gene panels. Compound heterozygous variants in MCM9 can cause POI, presenting with delayed puberty and primary amenorrhea in girls without a consanguineous family.

Homologous recombination deficiency gene panel analysis results in synchronous endometrial and ovarian cancers.

The objective of this study was to analyze the genetic alterations of tumors within the scope of the homologous recombination deficiency gene panel in patients diagnosed with synchronous endometrial ovarian cancer who have been followed for over 5 years using next-generation sequencing. DNA was isolated from the patient's formalin-fixed, paraffin-embedded tissue blocks. Next-generation sequencing was performed using the Illumina capture-based sequencing method. Samples were sequenced using the Sophia HR Solution DNA Kit. Seven patients were included in this study. The ratios of likely pathogenic (LP)/pathogenic (P) somatic mutations in ATM (serine/threonine kinase or Ataxia-telangiectasia mutated gene), BRCA2 (breast cancer type 2 susceptibility gene), BARD1 (BRCA1 associated RING domain 1), TP53 (tumor protein p53), BIRP1 (BRCA1-interacting helicase 1 gene), PALB2 (partner and localizer of BRCA2), and CHECK2 were 21 (48.8%), 8 (18.6%), 5 (11.6%), 3 (6.9%), 2 (4.6%), 2 (4.6%), and 2 (4.6%), respectively, in endometrium, and the ratios of somatic mutations in ATM, BRCA2, TP53, BARD1, RAD54L (DNA repair/recombination protein like), BIRP1, and RAD51D (RAD51 recombinase paralog D) were 24 (60%), 6 (15%), 5 (12.5%), 2 (5%), 2 (5%), 1 (2.5%), and 1 (2.5%), respectively, in ovary. In endometrioid-synchronous endometrial ovarian cancer cases, P/LP mutations were observed in ATM and CHECK2 genes in endometrium and ATM, BRCA2, and TP53 genes in ovary. In two non-endometrioid-synchronous endometrial ovarian cancer cases, CHEK2 (checkpoint kinase 2) mutations were observed in endometrium and ATM and TP53 mutations in ovary, whereas in one case, P/LP mutations in ATM and TP53 genes were common in both tissues. Pathogenic variations confirming the diagnosis of synchronous endometrial ovarian cancer with genetic alterations were identified in all but one case. ATM gene mutation emerged as the most common alteration and has a potential association with a favorable prognosis.

The High Diagnostic Yield of Prenatal Exome Sequencing Followed by 3400 Gene Panel Analysis in 629 Ongoing Pregnancies With Ultrasound Anomalies.

The aim of this study was to evaluate the diagnostic yield of routine exome sequencing (ES) in fetuses with ultrasound anomalies. We performed a retrospective analysis of the ES results of 629 fetuses with isolated or multiple anomalies referred in 2019-2022. Variants in a gene panel consisting of approximately 3400 genes associated with multiple congenital anomalies and/or intellectual disability were analyzed. We used trio analysis and filtering for de novo variants, compound heterozygous variants, homozygous variants, X-linked variants, variants in imprinted genes, and known pathogenic variants. Pathogenic and likely pathogenic variants (class five and four, respectively) were identified in 14.0% (88/629, 95% CI 11.5%-16.9%) of cases. In the current cohort, the probability of detecting a monogenetic disorder was ∼1:7 (88/629, 95% CI 1:8.7-1:5.9), ranging from 1:9 (49/424) in cases with one major anomaly to 1:5 (32/147) in cases with multiple system anomalies. Our results indicate that a notable number of fetuses (1:7) with ultrasound anomalies and a normal chromosomal microarray have a (likely) pathogenic variant that can be detected through prenatal ES. These results warrant implementation of exome sequencing in selected cases, including those with an isolated anomaly on prenatal ultrasound.

DIAGNOSTIC YIELD OF AN INHERITED RETINAL DISEASE GENE PANEL IN RETINOPATHY OF UNKNOWN ORIGIN.

Evaluating the presence of class 3, 4, and 5 genetic variants in inherited retinal disease (IRD) genes in patients with retinopathy of unknown origin (RUO). Multicentric retrospective study of RUO cases diagnosed between January 2012 and February 2022. General and ophthalmologic history, complete ophthalmologic examination, antiretinal antibodies, and IRD gene panel results were analyzed in every patient. Four RUO categories were defined: nonparaneoplastic autoimmune retinopathy, unilateral pigmentary retinopathy, asymmetrical pigmentary retinopathy, and acute zonal occult outer retinopathy. The authors included 12 patients (9 females) across these four RUO categories. Mean age at inclusion was 45.6 years (20-68 years). Seven patients demonstrated class 3 variants in IRD genes. Of these, two also demonstrated class 5 variants in other IRD genes. The remaining five patients had negative panel results. IRD gene panel analysis allowed diagnosis refinement in 1 (8.3%) nonparaneoplastic autoimmune retinopathy patient in the RUO cohort. When considering the nonparaneoplastic autoimmune retinopathy subpopulation only, a higher diagnostic yield of 20% (1/5 patients) was achieved. Every suspected nonparaneoplastic autoimmune retinopathy patient should benefit from gene panel testing to not overlook undiagnosed IRDs. By contrast, unilateral pigmentary retinopathy, asymmetrical pigmentary retinopathy, and acute zonal occult outer retinopathy subpopulations did not benefit from genetic testing in this study.

LZTR1 loss-of-function variants associated with café au lait macules with or without freckling.

Pathogenic variants in the leucine zipper-like transcriptional regulator 1 gene (LZTR1) have been identified in schwannomatosis and Noonan syndrome. Here, we expand the phenotype spectrum of LZTR1 variants. We identified four loss-of-function heterozygous LZTR1 variants in five children with multiple café au lait macules and one adult with multiple café au lait macules and axillar freckling, by applying gene panel analysis in four families. The three LZTR1 variants, namely, c.184del/p.Glu62Serfs*39, c.1927C < T/p.Gln643*, and c.857_858delinsT/p.Gly286Valfs*65, were novel, whereas the variant c.1018C > T/ p.Arg340* had been previously reported in a patient with schwannomatosis. Similar to what is known from other LZTR1-associated conditions, penetrance of the skin manifestations was reduced in two carriers of the familial variants. Our study expands the LZTR1 phenotype to the presence of isolated café au lait macules with or without freckling. Thus, variants in the LZTR1 gene should be considered in patients with multiple café au lait macules.

Mitochondrial DNA Depletion Syndromes Gene Panel versus Clinical Exome Sequencing in Children with Suspected Mitochondrial Hepatopathies

Introduction: Mitochondrial DNA depletion syndromes (MDDSs) are a group of clinically and genetically heterogeneous disorders. In the present study, we aimed to investigate the frequency of MDDS in children under the age of 5 years with suspected mitochondrial hepatopathy and to evaluate this group of patients using MDDS gene panel and clinical exome sequencing (CES) genetic analysis methods. Methods: Patients under 5 years of age who were clinically suspected to have mitochondrial hepatopathy and had neonatal acute liver failure, hepatic steatohepatitis, cholestasis, or cirrhosis with chronic liver failure of insidious onset were included. Results: Forty patients (20 female, 50%) were enrolled, with a median age of 102 [57–263.8] days. Icteric appearance was identified in 28 (70%) of the patients, hepatomegaly in 27 (67.5%), splenomegaly in 10 (25.0%), and hypotonicity in 10 (25.0%); moreover, elevated international normalized ratio was detected in 77.5%, cholestasis in 77.5%, and elevated lactate levels in 62.5%. Molecular genetic diagnosis was made in 9 patients (22.5%) with the MDDS gene panel and in 17 (42.5%) patients with the CES analysis. All patients diagnosed with MDDS had a history of parental consanguinity, while the rate in those without MDDS was 54.8% (p = 0.012). High lactate levels were identified in all those with MDDS, but in only 51.6% of those without MDDS (p = 0.020). Conclusion: Present study revealed that demographic findings and laboratory assessments are insufficient to diagnose genetically inherited diseases in children presenting with hepatic involvement. While one-fifth of the patients with suspected mitochondrial hepatopathies were diagnosed with MDDS, it is revealed that around half of patients can be diagnosed with CES panel.

Molecular genetic analysis of candidate genes for glutaric aciduria type II in a cohort of patients from Queensland, Australia

Glutaric aciduria type II (GAII) is a heterogeneous genetic disorder affecting mitochondrial fatty acid, amino acid and choline oxidation. Clinical manifestations vary across the lifespan and onset may occur at any time from the early neonatal period to advanced adulthood. Historically, some patients, in particular those with late onset disease, have experienced significant benefit from riboflavin supplementation. GAII has been considered an autosomal recessive condition caused by pathogenic variants in the gene encoding electron-transfer flavoprotein ubiquinone-oxidoreductase (ETFDH) or in the genes encoding electron-transfer flavoprotein subunits A and B (ETFA and ETFB respectively). Variants in genes involved in riboflavin metabolism have also been reported. However, in some patients, molecular analysis has failed to reveal diagnostic molecular results. In this study, we report the outcome of molecular analysis in 28 Australian patients across the lifespan, 10 paediatric and 18 adult, who had a diagnosis of glutaric aciduria type II based on both clinical and biochemical parameters. Whole genome sequencing was performed on 26 of the patients and two neonatal onset patients had targeted sequencing of candidate genes. The two patients who had targeted sequencing had biallelic pathogenic variants (in ETFA and ETFDH). None of the 26 patients whose whole genome was sequenced had biallelic variants in any of the primary candidate genes. Interestingly, nine of these patients (34.6%) had a monoallelic pathogenic or likely pathogenic variant in a single primary candidate gene and one patient (3.9%) had a monoallelic pathogenic or likely pathogenic variant in two separate genes within the same pathway. The frequencies of the damaging variants within ETFDH and FAD transporter gene SLC25A32 were significantly higher than expected when compared to the corresponding allele frequencies in the general population. The remaining 16 patients (61.5%) had no pathogenic or likely pathogenic variants in the candidate genes. Ten (56%) of the 18 adult patients were taking the selective serotonin reuptake inhibitor antidepressant sertraline, which has been shown to produce a GAII phenotype, and another two adults (11%) were taking a serotonin-norepinephrine reuptake inhibitor antidepressant, venlafaxine or duloxetine, which have a mechanism of action overlapping that of sertraline. Riboflavin deficiency can also mimic both the clinical and biochemical phenotype of GAII. Several patients on these antidepressants showed an initial response to riboflavin but then that response waned. These results suggest that the GAII phenotype can result from a complex interaction between monoallelic variants and the cellular environment. Whole genome or targeted gene panel analysis may not provide a clear molecular diagnosis.

Genetic Landscape of SH3TC2 variants in Russian patients with Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease type 4C (CMT4C) OMIM#601596 stands out as one of the most prevalent forms of recessive motor sensory neuropathy worldwide. This disorder results from biallelic pathogenic variants in the SH3TC2 gene. Within a cohort comprising 700 unrelated Russian patients diagnosed with Charcot-Marie-Tooth disease, we conducted a gene panel analysis encompassing 21 genes associated with hereditary neuropathies. Among the cohort, 394 individuals exhibited demyelinating motor and sensory neuropathy. Notably, 10 cases of CMT4C were identified within this cohort. The prevalence of CMT4C among Russian demyelinating CMT patients lacking the PMP22 duplication is estimated at 2.5%, significantly differing from observations in European populations. In total, 4 novel and 9 previously reported variants in the SH3TC2 gene were identified. No accumulation of a major variant was detected. Three previously reported variants, c.2860C>T p. (Arg954*), p. (Arg658Cys) and c.279G>A p. (Lys93Lys), recurrently detected in unrelated families. Nucleotide alteration p. (Arg954*) is present in most of our patients (30%).

#2316 Towards the new era of nephrology. What implications do genetic studies have on individuals with kidney disease?

Abstract Background and Aims The study of kidney disease (KD), including genetics, may involve a significant change in the management, treatment, and prognosis of the patient. Method Retrospective study assessing the impact of genetic studies (GS) requested by the nephrology department at Virgen Macarena Hospital over 43 months (June/2019 to December/2022). We analyze diagnoses before and after GS, as well as their impact. The sequencing platforms used include Illumina HiSeq-MiSeq-NovaSeq and Ion Torrent. Common databases consulted include ClinVar, RefSeq, Ensembl, NCBI, ExAC, aHUS, and BIER. Results A total of 118 patients with genetic studies (GS) were analyzed. The mean age was 45.2 ± 16, with 51% being male and 49% female. 78% had first and/or second-degree relatives with kidney disease, and in 43 cases (36%), no family history was known. The mean creatinine (Cr) was 1.9±2 mg/dl, glomerular filtration rate (GFR) was 67 ± 40 ml/min, and urine albumin/creatinine ratio (UAC) was 770 ± 1700 mg/g, with hematuria present in 57.6% of patients. A renal biopsy (RB) was performed in 32 patients (27%). The indications for genetic testing were as follows: extension of studies in biopsied nephropathy (20%), cystic kidney disease (29%), non-biopsiable or chronic kidney disease (21%), family history (33%), and for research or trial purposes (1%). In 22%, a study of a known familial mutation was conducted, while the remaining cases involved gene panel analysis. The pre-genetic diagnoses were as follows: 39% glomerular disease, 22% other conditions (suspected Alport and Fabry), 16% autosomal dominant polycystic kidney disease (ADPKD), 8% complement-associated pathology (CAP), 5% other cystic diseases, 4% tubulointerstitial nephropathies (TIN), 3% nephropathy of unknown origin, 2% diabetic nephropathy, and 2% nephroangiosclerosis. GS was positive in 76 patients (N = 76; 64%). Variants were detected in 76 patients, with 14 patients exhibiting two concurrent variants in the same or different genes, and 4 patients having up to 3 variants. The most frequent variants were identified in the genes: COL4A, PKD1, C3, MCP, and CFH. Of these, 37% were pathogenic mutations, 26.6% were likely pathogenic, 33% were variants of uncertain significance (VUS) possibly related to the pathology, and 3.2% were unrelated VUS. In cases with negative GS results (42; 35%), we considered that the pre-genetic diagnosis was excluded in 17% of the overall patient cohort. Post-GS diagnoses included Alport (30; 25%), ADPKD (16, 14%), CAP (13; 11%), focal segmental glomerulosclerosis (6; 5%), TIN (4; 3%), autosomal recessive polycystic kidney disease (ARPKD) (1; 1%), congenital anomalies of the kidney and urinary tract (CAKUT) (1; 1%), and other conditions (5; 4%). The concordance between the pre-genetic diagnosis and GS result was 52% (N = 61). It led to a change in diagnosis in 48% of patients (N = 56); both positive and exclusionary results had an impact on clinical management in 83% (N = 98), and it resulted in a change in treatment in 20% (N = 24). Conclusion Genetic studies allow us to trace the origin of the kidney disease, providing a fundamental diagnostic tool. According to the literature, the most frequently diagnosed variants were those related to Alport syndrome, renal polycystic disease (with PKD1 being the most common), and mutations associated with complement pathway pathology. The implications and diagnostic changes brought about by genetic studies justify their implementation. In some cases, they also reclassify the kidney disease in our patients, enabling access to specific treatments.

Comprehensive analysis of two hotspot codons in the TUBB4B gene and associated phenotypes

Our purpose was to elucidate the genotype and ophthalmological and audiological phenotype in TUBB4B-associated inherited retinal dystrophy (IRD) and sensorineural hearing loss (SNHL), and to model the effects of all possible amino acid substitutions at the hotspot codons Arg390 and Arg391. Six patients from five families with heterozygous missense variants in TUBB4B were included in this observational study. Ophthalmological testing included best-corrected visual acuity, fundus examination, optical coherence tomography, fundus autofluorescence imaging, and full-field electroretinography (ERG). Audiological examination included pure-tone and speech audiometry in adult patients and auditory brainstem response testing in a child. Genetic testing was performed by disease gene panel analysis based on genome sequencing. The molecular consequences of the substitutions of residues 390 and 391 on TUBB4B and its interaction with α-tubulin were predicted in silico on its three-dimensional structure obtained by homology modelling. Two independent patients had amino acid exchanges at position 391 (p.(Arg391His) or p.(Arg391Cys)) of the TUBB4B protein. Both had a distinct IRD phenotype with peripheral round yellowish lesions with pigmented spots and mild or moderate SNHL, respectively. Yet the phenotype was milder with a sectorial pattern of bone spicules in one patient, likely due to a genetically confirmed mosaicism for p.(Arg391His). Three patients were heterozygous for an amino acid exchange at position 390 (p.(Arg390Gln) or p.(Arg390Trp)) and presented with another distinct retinal phenotype with well demarcated pericentral retinitis pigmentosa. All showed SNHL ranging from mild to severe. One additional patient showed a variant distinct from codon 390 or 391 (p.(Tyr310His)), and presented with congenital profound hearing loss and reduced responses in ERG. Variants at codon positions 390 and 391 were predicted to decrease the structural stability of TUBB4B and its complex with α-tubulin, as well as the complex affinity. In conclusion, the twofold larger reduction in heterodimer affinity exhibited by Arg391 substitutions suggested an association with the more severe retinal phenotype, compared to the substitution at Arg390.

Two case reports of a novel missense mutation in the PNPLA6 gene in two siblings with chorioretinal dystrophy, hypogonadotropic hypogonadism, and cerebellar ataxia.

Boucher Neuhäuser Syndrome (BNS) is a rare disease with autosomal recessive inheritance defined by the classical triad; early-onset ataxia, hypogonadism and chorioretinal dystrophy. We present two siblings diagnosed with BNS at midlife, identified with homozygous state of a novel PNPLA6 missense mutation. One healthy sibling and the mother were heterozygous carriers of the mutation. The proband presented with the classical triad and the other sibling presented with visual problems at first. The proband was referred to our department by a private Neurologist, in early adulthood, because of hypogonadism, cerebellar ataxia, axonal neuropathy, and chorioretinal dystrophy for further evaluation. The sibling was referred to our department for evaluation, at childhood, due to visual problems. Later, the patient displayed the triad of ataxia, hypogonadotropic hypogonadism, and chorioretinal dystrophy. The unusual medical history of the two siblings led to further examinations and eventually the diagnosis of the first BNS cases in Cyprus. WES-based ataxia in silico gene panel analysis revealed 15 genetic variants and further filtering analysis revealed the PNPLA6 c.3323G > A variant. Segregation analysis in the family with Sanger sequencing confirmed the PNPLA6 homozygous variant c.3323G > A, p.Arg1108Gln in exon 29. This highlights the importance of considering rare inherited causes of visual loss, spinocerebellar ataxia, or/and HH in a neurology clinic and the significant role of genetic sequencing in the diagnostic process.

Analysis of CSF3R mutations in atypical chronic myeloid leukemia and other myeloid malignancies

We report a series of patients with CSF3R-mutant (CSF3Rmut) atypical chronic myeloid leukemia (aCML), chronic neutrophilic leukemia (CNL) or other hematologic malignancies. We included 25 patients: 5 aCML and 4 CNL CSF3Rmut patients; 1 aCML, 2 CNL, and 2 myelodysplastic/myeloproliferative neoplasm, not otherwise specified patients without CSF3R mutation; and 11 CSF3Rmut patients with other diseases [8 acute myeloid leukemia (AML), 1 chronic myelomonocytic leukemia (CMML), 1 myelodysplastic syndrome (MDS), and 1 acute lymphoblastic leukemia (ALL)]. Patients with aCML or CNL were tested by Sanger sequencing and pyrosequencing to identify CSF3R T618I. Twenty-two patients underwent gene panel analysis. CSF3R mutations, mostly T618I (8/9), were found at high frequencies in both aCML and CNL patients [5/6 aCML and 4/6 CNL]. Two aCML patients in early adulthood with CSF3R T618I and biallelic or homozygous CEBPA mutations without other mutations presented with increased blasts and exhibited remission for >6 years after transplantation. The other 7 CSF3Rmut aCML or CNL patients were elderly adults who all had ASXL1 mutations and frequently presented with SEBP1 and SRSF2 mutations. Five AML patients had CSF3R exon 14 or 15 point mutations, and 6 other patients (3 AML, 1 CMML, 1 MDS, and 1 ALL) had truncating mutations, demonstrating differences in leukocyte counts and mutation status. In conclusion, CSF3R mutations were found at a higher frequency in aCML patients than in previous studies, which might reflect ethnic differences. Additional studies are needed to confirm these findings and the relationship between CSF3R and CEBPA mutations.

A novel mitochondrial DNA variant in MT-ND6: m.14430A>C p.(Trp82Gly) identified in a patient with Leigh syndrome and complex I deficiency

Leigh syndrome is a severe progressive mitochondrial disorder mainly affecting children under the age of 5 years. It is caused by pathogenic variants in any one of more than 75 known genes in the nuclear or mitochondrial genomes.A 19-week-old male infant presented with lactic acidosis and encephalopathy following a 2-week history of irritability, neuroregression and poor weight gain. He was hypotonic with pathological reflexes, impaired vision, and nystagmus. Brain MRI showed extensive bilateral symmetrical T2 hyperintense lesions in basal ganglia, thalami, and brainstem. Metabolic workup showed elevated serum alanine, and heavy lactic aciduria with increased ketones, fumarate, malate, and alpha-ketoglutarate as well as reduced succinate on urine organic acid analysis. Lactic acidemia persisted, with only a marginally elevated lactate:pyruvate ratio (16.46, ref. 0–10). He demised at age 7 months due to respiratory failure.Exome sequencing followed by virtual gene panel analysis for pyruvate metabolism and mitochondrial defects could not identify any nuclear cause for Leigh syndrome. Mitochondrial DNA (mtDNA) genome sequencing revealed 88% heteroplasmy for a novel variant, NC_012920.1(MT-ND6):m.14430A>C p.(Trp82Gly), in blood DNA. This variant was absent from the unaffected mother's blood, fibroblast, and urine DNA, and detected at a level of 5% in her muscle DNA.Mitochondrial respiratory chain analysis revealed markedly reduced mitochondrial complex I activity in patient fibroblasts (34% of parent and control cells), and reduced NADH-linked respirometry (less than half of parental and control cells), while complex II driven respirometry remained intact. The combined clinical, genetic, and biochemical findings suggest that the novel MT-ND6 variant is the likely cause of Leigh syndrome in this patient. The mitochondrial ND6 protein is a subunit of complex I.An interesting finding was the absence of a significantly elevated lactate:pyruvate ratio in the presence of severe lactatemia, which directed initial diagnostic efforts towards excluding a pyruvate metabolism defect. This case highlights the value of a multidisciplinary approach and complete genetic workup to diagnosing mitochondrial disorders in South African patients.

Genotype-phenotype of autosomal dominant polycystic kidney disease in Malta

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple renal cysts causing kidney enlargement and end-stage renal disease (ESRD) in half the patients by 60 years of age. The aim of the study was to determine the genetic aetiology in Maltese patients clinically diagnosed with ADPKD and correlate the clinical features. MethodsA total of 60 patients over 18 years of age clinically diagnosed with ADPKD were studied using a customized panel of genes that had sufficient evidence of disease diagnosis using next generation sequencing (NGS). The genes studied were PKD1, PKD2, GANAB, DNAJB11, PKHD1 and DZIP1L. Selected variants were confirmed by bidirectional Sanger sequencing with specifically designed primers. Cases where no clinically significant variant was identified by the customized gene panel were then studied by Whole Exome Sequencing (WES). Microsatellite analysis was performed to determine the origin of an identified recurrent variant in the PKD2 gene. Clinical features were studied for statistical correlation with genetic results. ResultsGenetic diagnosis was reached in 49 (82%) of cases studied. Pathogenic/likely pathogenic variants PKD1 and PKD2 gene were found in 25 and in 23 cases respectively. The relative proportion of genetically diagnosed PKD1:PKD2 cases was 42:38. A pathogenic variant in the GANAB gene was identified in 1 (2%) case. A potentially significant heterozygous likely pathogenic variant was identified in PKHD1 in 1 (2%) case. Potentially significant variants of uncertain significance were seen in 4 (7%) cases of the study cohort. No variants in DNAJB11 and DZIP1L were observed. Whole exome sequencing (WES) added the diagnostic yield by 10% over the gene panel analysis. Overall no clinically significant variant was detected in 6 (10%) cases of the study population by a customized gene panel and WES. One recurrent variant the PKD2 c.709+1G > A was observed in 19 (32%) cases. Microsatellite analysis showed that all variant cases shared the same haplotype indicating that their families may have originated from a common ancestor and confirmed it to be a founder variant in the Maltese population.The rate of decline in eGFR was steeper and progression to ESRD was earlier in cases with PKD1 variants when compared to cases with PKD2 variants. Cases segregating truncating variants in PKD1 showed a significantly earlier onset of ESRD and this was significantly worse in cases with frameshift variants. Overall extrarenal manifestations were commoner in cases segregating truncating variants in PKD1. ConclusionsThis study helps to show that a customized gene panel is the first-line method of choice for studying patients with ADPKD followed by WES which increased the detection of variants present in the PKD1 pseudogene region. A founder variant in the PKD2 gene was identified in our Maltese cohort with ADPKD. Phenotype of patients with ADPKD is significantly related to the genotype confirming the important role of molecular investigations in the diagnosis and prognosis of polycystic kidney disease. Moreover, the findings also highlight the variability in the clinical phenotype and indicate that other factors including epigenetic and environmental maybe be important determinants in Autosomal Dominant Polycystic Kidney Disease.

Abstract 7507: Capturing live single circulating tumor cells platform for comprehensive genomic profiling with paired ctDNA in breast cancer patients

Abstract Background: Single circulating tumor cells (sCTC) capture without cell fixation with no blood contaminants like leukocytes are highly challenging. sCTC genome transcriptomics anticipates new avenues in new discovery, targeted therapies, and inclusion of patients in clinical trials where the tissue is inaccessible. We report, an assay to selectively capture live sCTCs at singularity with NGS-based comprehensive mutational profiling in breast cancer (BC) patients. Further, paired ctDNA outcomes using blood with mutational heterogeneity scores of sCTCs adds comprehensive genomics for establishing an accelerated treatment outcomes and resistance signatures. Methods: Retrospectively, sCTC were isolated from 70 breast cancer (BC) patients using OncoRadar platform consisting affinity-based glass beads mediated by anti EpCAM antibody. CTCs were selectively captured and released without fixing and contamination of any leukocytes. Platform possess cleavable disulfide spacer at pH 8.0, between glass beads (diam. 2 mm, area 12.57 mm2) and a ligand. Sensitivity, specificity, positive predictive value (PPV), negative prediction value (NPV), and accuracy were evaluated using MCF-7 cells and CTCs. Assay to isolate CTC has been developed in 96 well plate, for downstream NGS-based comprehensive gene panel analysis using hybridization-based target enrichment. Interestingly, no mutations detected in blood samples (~20%) for CtDNA versus CTC DNA offered non-chip implicated mutations. Results:. Analytical validation using OncoRadar showed higher cell capture efficiency (99%) with spiked MCF-7 cells/ml of blood. Sensitivity and specificity values were 97% and 98% respect, while PPV was 99% and NPV was 7.5% with the accuracy parameter of 99%. Sensitivity, specificity, PPV, and NPV parameters when assessed for CTC isolation from 70 BC patients. NGS-based mutational analysis, sCTC showed the presence of PIK3CA, ESR1, and NF1 mutations, which were absent from paired ctDNA samples. Conclusion: Detection of ctDNA obscure mutations in paired sCTC samples suggests high sensitivity of sCTC-based genomic assays compared to current standard of practice. Heterogeneity scores amongst inter and intra CTCs with MATH scores offer new dimensions in genomics. Combination of ctDNA and sCTC DNA assay offers a comprehensive realistic genomic outcome for more significant therapy decisions, especially when guideline based options may exhausts. Citation Format: Gowhar Shafi, Atul Bharde, Ganesh Khutale, Saloni Andhari, Richa Deshpande, Bhagwat Jadhav, Sangeeta Prajapati, Moubeen Fauzul, Kanchan Hariramani, Madhura Basavalingegowda, Jayant Khandare. Capturing live single circulating tumor cells platform for comprehensive genomic profiling with paired ctDNA in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7507.