Partial Gene Deletions Research Articles

5159 Bone Disease Related To PLS3 Gene Mutation

Abstract Disclosure: S. Saberi: None. N. Esfandiari: None. Background: A variety of genes play a role in the development of osteoporosis. One of these is PLS3, located on the X chromosome. PLS3 gene mutation leads to defective function of the protein Plastin 3. Plastin 3 functions in the binding and bundling of F-actin, a main component of the actin cytoskeleton. In the skeletal system the actin cytoskeleton regulates adaption to gravity and mechanical stress. Dysregulation of this gene is associated with diseases of the bone, including osteoporosis. Clinical Case: A 60-year-old man with a history of hearing loss since age 52, hypothyroidism diagnosed at age 57 presents to the Genetics Clinic after 2 grandsons were diagnosed with osteogenesis imperfecta. The mothers (his two daughters) of the two grandsons were found to be carriers of the PLS3 gene mutation. Because of their carrier status the patient was screened and a pathogenic partial gene deletion of exons 8 to 16 in the PLS3 gene was detected. The patient has a history of a right wrist fracture at age 16 in a fall from 8 feet height from a ladder, right leg fracture at age 45 when his leg got caught and twisted in a ladder rung, 5 rib fractures and shoulder fracture at age 53 in a fall from 3 feet into a utility hole. A DXA scan showed osteopenia in his left femoral neck (T-score -1.3), normal bone density in his total hip (T score -0.1) and lumbar spine (L1-L4 T-score -0.4). FRAX score for major osteoporotic fracture was 9.4% and hip fracture was 1.2%. Labs for secondary causes of osteoporosis were normal. Due to his gene mutation and fracture history a bisphosphonate was recommended.Clinical Lessons:Obtaining a thorough family history in people with fractures can determine the need for screening for gene mutations associated with osteoporosis. This can identify patients who need more aggressive management. Presentation: 6/2/2024

Deletions in the CDKL5 5' untranslated region lead to CDKL5 deficiency disorder.

Pathogenic variants in the cyclin-dependent kinase-like 5 (CDKL5) gene are associated with CDKL5 deficiency disorder (CDD), a severe X-linked developmental and epileptic encephalopathy. Deletions affecting the 5' untranslated region (UTR) of CDKL5, which involve the noncoding exon 1 and/or alternatively spliced first exons (exons 1a-e), are uncommonly reported. We describe genetic and phenotypic characteristics for 15 individuals with CDKL5 partial gene deletions affecting the 5' UTR. All individuals presented characteristic features of CDD, including medically refractory infantile-onset epilepsy, global developmental delay, and visual impairment. We performed RNA sequencing on fibroblast samples from three individuals with small deletions involving exons 1 and/or 1a/1b only. Results demonstrated reduced CDKL5 mRNA expression with no evidence of expression from alternatively spliced first exons. Our study broadens the genotypic spectrum for CDD by adding to existing evidence that deletions affecting the 5' UTR of the CDKL5 gene are associated with the disorder. We propose that smaller 5' UTR deletions may require additional molecular testing approaches such as RNA sequencing to determine pathogenicity.

Atypical noncontiguous TSC2/PKD1 gene deletions presenting as tuberous sclerosis/polycystic kidney disease contiguous gene syndrome.

Tuberous sclerosis complex (TSC) and autosomal dominant polycystic kidney disease (ADPKD) are genetically distinct disorders typically associated with pathogenic variants in TSC1 and TSC2 for the former and PKD1 and PKD2 for the latter. TSC2 and PKD1 lie adjacent to each other, and large deletions comprising both genes lead to TSC2/PKD1 contiguous gene deletion syndrome (CGS). In this study, we describe a young female patient exhibiting symptoms of TSC2/PKD1 CGS in which genetic analysis disclosed two noncontiguous partial gene deletions in TSC2 and PKD1 that putatively are responsible for the manifestations of the syndrome. Further analysis revealed that both deletions appear to be de novo on the maternal chromosome, presumably with a germline origin. Despite extensive analysis, no maternal chromosomal rearrangement triggering these pathogenic variants was detected. This case elucidates a unique pathogenesis for TSC2/PKD1 CGS, diverging from the common contiguous deletions typically observed, marking the first reported instance of TSC2/PKD1 CGS caused by independent, functionally significant partial gene deletions.

IGF1 Haploinsufficiency: Phenotype and Response to Growth Hormone Treatment in 9 Patients.

The clinical features of bi-allelic IGF1 defects are well established, i.e., severe growth failure and microcephaly, delayed psychomotor development, and sensorineural deafness. However, information on clinical and endocrine consequences of heterozygous IGF1 variants and treatment options is scarce. We aimed at extending the knowledge base of the clinical presentation and growth response to recombinant human growth hormone (rhGH) of patients carrying such variants. Retrospective case series of patients with pathogenic heterozygous IGF1 variants. Nine patients from six families were included, harbouring five whole or partial gene deletions and one frameshift variant resulting in a premature stop codon (three de novo, one unknown inheritance). In the other two families, variants segregated with short stature. Mean (SD) birth length was -1.9 (1.3) SDS (n = 7), height -3.8 (0.6) SDS, head circumference -2.5 (0.6) SDS, serum IGF-I -1.9 (0.7) SDS, serum IGFBP-3 1.1 (0.4) SDS (n = 7), and GH peak range 5-31 μg/L (n = 4). Five patients showed feeding problems in infancy. Average height increased after 1 and 2 years of rhGH treatment by 0.8 SDS (range 0.3-1.3 SDS) and 1.3 SDS (range 0.5-2.0 SDS), respectively. Adult height in 2 patients was -2.8 and -1.3 SDS, which was, respectively, 1.3 and 2.9 SDS taller than predicted before start of treatment. Haploinsufficiency of IGF1 causes a variable phenotype of prenatal and postnatal growth failure, microcephaly, feeding difficulties, low/low-normal serum IGF-I values in contrast to serum IGFBP-3 in the upper-normal range. Treatment with rhGH increased growth in the first 2 years of treatment, and in 2 patients adult height after treatment was higher than predicted at treatment initiation.

Mild phenotypes in patients with different deletions in the 3' enhancer region of SHOX.

Haploinsufficiency of the short stature homeobox-containing (SHOX) gene leads to a phenotypic spectrum ranging from Leri-Weill dyschondrosteosis (LWD) to SHOX-deficient short stature. SHOX nullizygosity leads to Langer mesomelic dysplasia. Pathogenic variants can include whole or partial gene deletions or duplications, point mutations within the coding sequence, and deletions of upstream and downstream regulatory elements. Here we report two families: a non-consanguineous family with a deletion downstream of SHOX, in which the homozygous proband presented with isolated Madelung deformity, without LWD or short stature, as well as a 9-year-old girl with Madelung deformities, mesomelia, a dominant family history of Madelung deformity and a heterozygous deletion of the CNE9 region in the 3' downstream region of SHOX. These case reports provide additional information on the effects of 3' downstream deletions of SHOX, by demonstrating the limited phenotype associated with the recurrent 47.5 kb deletion in a homozygous state and the CNE9 deletion in a heterozygous state.

Structural rearrangements as a recurrent pathogenic mechanism for SETBP1 haploinsufficiency

Chromosomal structural rearrangements consist of anomalies in genomic architecture that may or may not be associated with genetic material gain and loss. Evaluating the precise breakpoint is crucial from a diagnostic point of view, highlighting possible gene disruption and addressing to appropriate genotype–phenotype association. Structural rearrangements can either occur randomly within the genome or present with a recurrence, mainly due to peculiar genomic features of the surrounding regions. We report about three non-related individuals, harboring chromosomal structural rearrangements interrupting SETBP1, leading to gene haploinsufficiency. Two out of them resulted negative to Chromosomal Microarray Analysis (CMA), being the rearrangement balanced at a microarray resolution. The third one, presenting with a complex three-chromosome rearrangement, had been previously diagnosed with SETBP1 haploinsufficiency due to a partial gene deletion at one of the chromosomal breakpoints. We thoroughly characterized the rearrangements by means of Optical Genome Mapping (OGM) and Whole Genome Sequencing (WGS), providing details about the involved sequences and the underlying mechanisms. We propose structural variants as a recurrent event in SETBP1 haploinsufficiency, which may be overlooked by laboratory routine genomic analyses (CMA and Whole Exome Sequencing) or only partially determined when associated with genomic losses at breakpoints. We finally introduce a possible role of SETBP1 in a Noonan-like phenotype.

Short-read whole genome sequencing identifies causative variants in most individuals with previously unexplained aniridia

BackgroundClassic aniridia is a highly penetrant autosomal dominant disorder characterised by congenital absence of the iris, foveal hypoplasia, optic disc anomalies and progressive opacification of the cornea. >90% of cases...

Identification of two novel large deletions in FBN1 gene by next-generation sequencing and multiplex ligation-dependent probe amplification.

Mutations in fibrillin-1 (FBN1) are known to be associated with Marfan syndrome (MFS), an autosomal dominant connective tissue disorder. Most FBN1 mutations are missense or nonsense mutations. Traditional molecular genetic testing for the FBN1 gene, like Sanger sequencing, may miss disease-causing mutations in the gene's regulatory regions or non-coding sequences, as well as partial or complete gene deletions and duplications. Next-generation sequencing, multiplex ligation-dependent probe amplification and gap PCR were conducted on two MFS patients to screen for disease-causing mutations. We identified two large deletions in FBN1 from two MFS patients. One patient had a 0.23 Mb deletion (NC_000015.9:g.48550506_48779360del) including 5'UTR-exon6 of FBN1. The other patient harbored a 1416 bp deletion (NC_000015.9:g.48410869_48412284del) affecting the last exon, exon 66, of the FBN1 gene. Our results expanded the number of large FBN1 deletions and highlighted the importance of screening for large deletions in FBN1 in clinical genetic testing, especially for those with the classic MFS phenotype.

Congenital myasthenic syndrome type 2C in a neonate: Redefining the phenotype of CHRNB1‐related myasthenic syndromes

AbstractObjectiveWe present a neonate with generalized weakness due to autosomal recessive congenital myasthenic syndrome type 2C (CMS2C) resulting from a compound heterozygous mutation in the CHRNB1 gene.Patient descriptionOur patient was determined by multiple methodologies to have a diagnosis of CMS2C (OMIM #616314). Whole‐genome sequencing revealed two distinct variants in the CHRNB1 gene (OMIM *100710): a maternally inherited 2 kb pathogenic microdeletion on chromosome 17p13.1 and a paternally inherited intronic deletion (c.1218‐9_1218‐7) that was reported by the laboratory as a variant of unknown significance.ConclusionsCMS2C is a rare autosomal recessive genetic condition associated with early‐onset muscle weakness. Our patient had a paternally inherited deletion in CHRNB1 (c.1218‐9_1218‐7) that was initially described as a variant of unknown significance. We suggest this finding is “likely pathogenic,” as this aberration has not been commonly described. He also had a partial deletion of CHRNB1 in the maternally inherited allele, which provides further evidence that partial gene deletions may be a more common molecular mechanism than previously known for this condition. The combination of the clinical presentation and electrophysiologic data allowed us to understand the molecular findings and ultimately diagnose CMS2C in our patient.

Cognitive, adaptive and behavioral profile in Sotos syndrome children with 5q35 microdeletion or intragenic variants.

Sotos syndrome (SoS) is a congenital overgrowth syndrome with variable degree of intellectual disability caused in the 90% of cases by pathogenetic variants of the Nuclear receptor binding SET Domain protein1 (NSD1) gene. NSD1 gene functions can be abrogated by different genetic alterations (i.e., small intragenic pathogenic variants like deletions/insertions, nonsense/missense pathogenic variants, partial gene deletions and whole deletions or microdeletion of 5q35 chromosomal region). Therefore, correlation of the genotype-phenotype with a possible contribution of more implicated genes to the medical, cognitive and behavioral profile is a topic of great interest. Although a more severe learning disability has been described in individuals with 5q35 microdeletion when compared to individuals with NSD1 intragenic pathogenic variants a fully delineated cognitive and behavioral phenotype has not been described yet. The importance of providing clinical characterization in relation to the genotype comes from the necessity to early identify children more at risk of developing psychopathological disorders. We characterize the cognitive, adaptive and behavioral phenotype of a pediatric sample of 64 individuals affected by SoS, performing a standardized neuropsychological evaluation. Secondly, we compare cognitive-behavioral profiles of SoS individuals carrying and not carrying the 5q35 microdeletion. SoS participants were characterized by a mild cognitive impairment of both Intellectual Quotient and adaptive skills in association to borderline symptoms of attention deficit. Our results suggest that the 5q35 microdeletion is associated with lower scores specifically concerning the cognitive, adaptive functioning and behavioral domains. However, longitudinal studies are necessary to confirm these findings and delineate a developmental trajectory of SoS.

Molecular Analysis and Reclassification of NSD1 Gene Variants in a Cohort of Patients with Clinical Suspicion of Sotos Syndrome.

Sotos syndrome is a rare genetic disorder caused by haploinsufficiency of the NSD1 (nuclear receptor binding SET domain containing protein 1) gene. No clinical diagnostic consensus criteria are published yet, and molecular analysis reduces the clinical diagnostic uncertainty. We screened 1530 unrelated patients enrolled from 2003 to 2021 at Galliera Hospital and Gaslini Institute in Genoa. NSD1 variants were identified in 292 patients including nine partial gene deletions, 13 microdeletions of the entire NSD1 gene, and 115 novel intragenic variants never previously described. Thirty-two variants of uncertain significance (VUS) out of 115 identified were re-classified. Twenty-five missense NSD1 VUS (25/32, 78.1%) changed class to likely pathogenic or likely benign, showing a highly significant shift in class (p < 0.01). Apart from NSD1, we identified variants in additional genes (NFIX, PTEN, EZH2, TCF20, BRWD3, PPP2R5D) in nine patients analyzed by the NGS custom panel. We describe the evolution of diagnostic techniques in our laboratory to ascertain molecular diagnosis, the identification of 115 new variants, and the re-classification of 25 VUS in NSD1. We underline the utility of sharing variant classification and the need to improve communication between the laboratory staff and the referring physician.

Applying CRISPR-Cas9 Genome Editing to Study Genes Involved in Peroxisome Biogenesis or Peroxisomal Functions.

The development and application of the CRISPR-Cas9 technology for genome editing of mammalian cells have opened up a wealth of possibilities for genetically modifying and manipulating human cells, and use in functional studies or therapeutic approaches.Here we describe the approach that we have been using successfully to generate multiple human cell lines with targeted (partial) gene deletions, i.e., knockout cells, or human cells with modified genomic nucleotide sequences, i.e., knock-in cells, in genes encoding known or putative proteins involved in peroxisome biogenesis or peroxisomal functions.

IKZF1 Deletions Are Markers of Treatment Resistance in Adult Ph-Negative B-Cell Acute Lymphoblastic Leukemia Patients Treated within the Ongoing Risk-Adapted Pethema LAL19 Trial

Introduction IKZF1 (Ikaros) deletion has been related with poor outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL). However, its impact in adult patients (pts) treated with MRD-oriented trials has not been fully established. This study aimed to assess if IKZF1 deletions are markers of poor prognosis in adults with Ph-negative BCP ALL treated within the ongoing risk-adapted PETHEMA LAL19 trial (NCT04179929). The impact of IKZF1 loss in these pts and those from the EWALL Group (European Working group on ALL) is being evaluated in a HARMONY (Healthcare Alliance for Resourceful Medicine Offensive against Neoplasms in Hematology) project (Project Ref ALL2). Methods In the PETHEMA LAL19 trial, pts with poor end-induction MRD clearance (MRD>0.01%) or high-risk genetics (low-hypodiploid, KMT2Ar, TP53 biallelic alteration or IKZF1+CDKN2A/B deletion) are assigned to allogeneic stem cell transplantation, the remaining pts receive maintenance chemotherapy. All pts are analyzed by G-banding, FISH, NGS (DNA gene panel) and SNP array. Next generation flow cytometry is used for MRD assessment. Main outcome measures: i) Complete Remission (CR): absence of extramedullary disease, neutrophil level >1x109/L, platelet level >100x109/L, and bone marrow blast level < 5%; ii) Cumulative Incidence of Relapse (CIR) and non-relapse mortality (NRM): calculated using cumulative incidence rates to accommodate competing risks and compared by Gray's test; iii) Overall survival (OS): measured from the time of protocol entry to the time of death or last contact. Results 147pts were analyzed. Mean age 40y [range 18-60], 56% males, 10% CNS involvement, 78% common B, 15% pro-B and 7% pre-B ALL. Ph-like (12%) was the most common genetic subtype followed by KMT2Ar (11%), PAX5 P80R (8%), low-hypodiploid (7%), TCF3::PBX1 and high-hyperdiploid (6% each), iAMP21 and ETV6::RUNX1 (2% each). More uncommon biological entities such as ZNF384r, MEF2Dr, MYCr, IDH1 R132C and IDH2 R140Q were seen in single pts (<1% each).The remaining patients (41%) were B-other,no growth or normal karyotype. IKZF1 deletion (IK DEL) was observed in 66/147(45%) pts. Deletions involved the whole gene (37/66, 56%) or a subset of exons (partial gene deletions). The most recurrent partial gene deletions (n=29) were: loss of exons 4-7 (n=6, 21%), 4-8 (n=5, 17%), 2-8 and 1-7 (n=4, 14% each), 1-3 (n=3, 10%), 2&3 and 2-7 (n=2, 7% each). The IKZF1plus profile was observed in 37/147 (25%) pts. IK DEL were more frequent in low-hypodiploid (100%) and Ph-like ALL (88%), and less prevalent in KMT2Ar (19%), PAX5 P80R (18%) and TCF3::PBX1 ALL (11%), p<0.001. Median follow up was 11.4 months. IKDEL pts showed lower CR rate than pts without Ikaros loss (75% vs. 91%, p=0.027). The IKZF1plus signature showed similar CR rate to IK DEL without IKZF1plus profile (IKZF1not plus) (73% vs. 78%, p=ns). IKDEL pts showed poorer MRD clearance than WT individuals. Interestingly, this fact was mainly attributable to partial gene deletions (MRD≥0.01% partial 79% vs. whole 42% vs. WT 29%, p=0.003). IKZF1plus was also correlated with positive MRD, however IKZF1not plus pts showed higher frequency of MRD positivity than IKZF1plus pts (MRD≥0.01% plus 47% vs. not plus 71% vs. WT 29%, p=0.012). Pts with whole gene loss showed lower OS than pts with partial gene losses or IKZF1 WT (1y-OS 62% vs. 88% vs. 89%, p=0.012)(Figure 1A). Of note, relapse was not the main cause of death of IK DEL whole gene pts (n=26) as only 1 patient died due to disease progression. Instead, NRM was the main cause of death in whole gene-deleted pts (4 died in Ind-1, 3 in Ind-2, 1 due to TRM and 1 due to Covid19 infection). Accordingly, the 1y-CIR of IK DEL whole pts was lower than that of IKZF1 WT individuals, who showed similar CIR to IK DEL partial ones (1y-CIR whole 7% vs. WT 30% vs. partial 32%, p=0.059) (Figure 1B). Pts with IKZF1plus signature did not show differences in OS and CIR compared to IKZF1 WT or IKZF1not plus individuals (data not shown). Conclusions This preliminary data suggest that IK DEL may be marker of poor treatment response in adult Ph-negative BCP ALL. Further analyses including all patients of the HARMONY project may identify which isoforms or in which genetic ALL subtypes IKZF1 DEL are particularly deleterious. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

RF27 | LBSUN361 Understanding The Genetics Of Multiple Endocrine Neoplasia Type 1: The Road Ahead

Abstract Background About 10-30% patients have genotype-negative (GN)-MEN1. Here, we compare our cohort of comprehensively phenotyped GN-MEN1 and genotype-positive (GP)-MEN1 patients. We also investigate somatic mosaicism as a cause of GN-MEN1. Methods GN-MEN1 patients showed no mutations in MEN1 or other primary hyperparathyroidism (PHPT) genes (CASR, RET, CDKN1B, CDC73, AIP, GNA11, AP2S1, GCM2) performed per clinical indication. Index patients (first patient in kindred to be diagnosed with MEN1) or probands (first patient in kindred to be evaluated at our center) from kindreds with GP-MEN1 were identified. All patients underwent testing for tumor biomarkers, imaging and evaluation for Zollinger-Ellison syndrome (ZES). Whole exome sequencing was performed on multiple tumors from 13 GN-MEN1 patients. GP-MEN1 patients were also categorized into high-impact (nonsense, frameshift, splice site, whole or partial gene deletion) or low-impact (missense or in-frame indels) mutation-bearing groups. Results We identified 43 (31 females, 12 males) patients with GN-MEN1 and 160 (102 index, 58 probands) patients with GP-MEN1 (98 females, 62 males). Among GP-MEN1, 97 had high impact and 34 had low-impact mutations. Mean age at last follow-up was 56 (±14) years for GN-MEN1 and 52 (±16) years for GP-MEN1 patients. Among the GN-MEN1 patients, 38/42 developed PHPT - 3/38 (8%) had recurrent disease. In GP-MEN1, 144/149 developed PHPT - 83/144 (58%) with recurrent disease. Median age of index presentation with PHPT significantly differed between the groups (27.5 years in GP-MEN1 vs 53 years in GN-MEN1 (χ2 (1)=5.5: p &amp;lt;0.05)). 33/42 GN-MEN1 patients had a pituitary adenoma (22/33 functioning - 9/22 prolactinomas; 8/22 GH-secreting). In comparison, 94/145 GP-MEN1 patients developed a pituitary adenoma (51 functioning - 37/51 prolactinomas, no GH-secreting tumors). In the GN-MEN1 group, 12/42 (29%) patients developed a gastro-entero-pancreatic (GEP)-NET. In comparison, 86/130 (66%) patients with GP-MEN1 had a GEP-NET. Somatic variants in MEN1, CDC73 or CDKI were observed in six tumors. However, these mutations were not seen in another tumor/s or germline DNA from the patient. All patients with high-impact mutations had PHPT (63% had recurrent disease), 70% had pituitary tumors (35% with macro-adenomas) and 61% developed GEP-NETs (29% with distant metastases). In comparison, 91% patients with low-impact mutations developed PHPT (41% with recurrent disease), 69% developed pituitary tumors (23% with macro-adenomas) and 65% developed GEP-NETs (35% with distant metastases). Conclusion Patients with GP-MEN1 generally experience younger age of onset, greater likelihood of recurrent PHPT and co-occurrence of GEP-NETs in comparison to GN-MEN1. Our findings do not support somatic mosaicism as a cause of GN-MEN1. No apparent correlation in phenotype between high-impact vs. low-impact mutations is noted. Presentation: Monday, June 13, 2022 12:51 p.m. - 12:56 p.m.

Genomic Aberrations Generate Fusion Gene FOXK2::TP63 and Activate NFKB1 in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a severe lymphoid malignancy with a worse prognosis lacking curative treatment regimens. Several gene mutations and deregulated pathways, including NFkB signaling, have been implicated in its pathogenesis. Accordingly, CTCL cell line HUT-78 reportedly contains mutated NFKB2, which is constitutively activated via partial gene deletion, also demonstrating that genomic rearrangements cause driving mutations in this malignancy. Here, along with HUT-78, we analyzed CTCL cell line HH to identify additional aberrations underlying gene deregulation. Karyotyping and genomic profiling of HH showed several rearrangements worthy of detailed investigation. Corresponding to the established karyotype, RNA-seq data and PCR analysis confirmed the presence of t(3;17)(q28;q25), generating a novel fusion gene, FOXK2::TP63. Furthermore, chromosomal rearrangement t(1;4)(p32;q25) was connected to amplification at 4q24–26, affecting aberrant NFKB1 overexpression thereat. Transcription factor binding-site analysis and knockdown experiments demonstrated that IRF4 contributed to NFKB1 expression. Within the same amplicon, we identified amplification and overexpression of NFkB signaling activator CAMK2D (4q26) and p53-inhibitor UBE2D3 (4q24). Genomic profiling data for HUT-78 detailed a deletion at 10q25 underlying reported NFKB2 activation. Moreover, amplifications of ID1 (20q11) and IKZF2 (2q34) in this cell line drove overexpression of these NK cell differentiation factors and possibly thus formed corresponding lineage characteristics. Target gene analysis for NFKB1 via siRNA-mediated knockdown in HH revealed activation of TP63, MIR155, and NOTCH pathway component RBPJ. Finally, treatment of HH with NFkB inhibitor demonstrated a role for NFkB in supporting proliferation, while usage of inhibitor DAPT showed significant survival effects via the NOTCH pathway. Collectively, our data suggest that NFkB and/or NOTCH inhibitors may represent reasonable treatment options for subsets of CTCL patients.

Pfhrp2/3 gene deletion and genetic variation in PfHRP2-based RDTs with P. falciparum positive samples from India and its implication on malaria control.

Recent studies have documented Pfhrp2/3 gene deletion globally as one of the biological threats in the fight against malaria. For malaria diagnosis, PfHRP2 based RDTs are most widely used in India, and performance of these RDTs are affected by deleted Pfhrp2/3 gene in Plasmodium falciparum. This study was planned to confirm Pfhrp2/3 gene deletion incidences and genetic variation in PfHRP2-based RDT positive with P.falciparum malaria cases from India. Confirmed positive samples by PfHRP2-based RDTs as P. falciparum (n=240) from six different endemic regions of India were validated by PCR to assure the actual infection. Two hundred forty samples qualified for DNA intactness by single-copy genes were subjected to amplification for the Pfhrp2/3 gene and its neighbouring gene (downstream and upstream) by PCR genotyping. Genetic variation in samples was analysed post-sequencing using Mega X software. Statistical analysis was performed to validate the genetic variation using Mann-Whitney Test. RDT target region of Pfhrp2 gene (exon2) was found deleted in a single sample with presence of the Pfhrp3 exon2. Complete gene deletion of 4.2% was observed in the Pfhrp3 gene. Partial gene deletion was recorded for both pfhrp2 gene (exon2-0.4%, upstream 25.8% and downstream -9.1%) and Pfhrp3 gene (exon2-18.75%, upstream - 22.08% and downstream 13.3%). Eleven new unique types of amino acid repeat sequence and earlier reported amino acid repeat type was found in the Pfhrp2 gene, prompting high genetic variation. This study suggests that parasites lacking Pfhrp2/3 gene and its neighbouring gene (downstream and upstream) are present in malaria endemic areas of India, resulting in false positive results by RDT. Systematic countrywide monitoring for malaria control and elimination of malaria is warranted in this regard.

Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss.

Inactivating variants as well as a missense variant in the centrosomal CEP78 gene have been identified in autosomal recessive cone-rod dystrophy with hearing loss (CRDHL), a rare syndromic inherited retinal disease distinct from Usher syndrome. Apart from this, a complex structural variant (SV) implicating CEP78 has been reported in CRDHL. Here we aimed to expand the genetic architecture of typical CRDHL by the identification of complex SVs of the CEP78 region and characterization of their underlying mechanisms. Approaches used for the identification of the SVs are shallow whole-genome sequencing (sWGS) combined with quantitative polymerase chain reaction (PCR) and long-range PCR, or ExomeDepth analysis on whole-exome sequencing (WES) data. Targeted or whole-genome nanopore long-read sequencing (LRS) was used to delineate breakpoint junctions at the nucleotide level. For all SVs cases, the effect of the SVs on CEP78 expression was assessed using quantitative PCR on patient-derived RNA. Apart from two novel canonical CEP78 splice variants and a frameshifting single-nucleotide variant (SNV), two SVs affecting CEP78 were identified in three unrelated individuals with CRDHL: a heterozygous total gene deletion of 235 kb and a partial gene deletion of 15 kb in a heterozygous and homozygous state, respectively. Assessment of the molecular consequences of the SVs on patient’s materials displayed a loss-of-function effect. Delineation and characterization of the 15-kb deletion using targeted LRS revealed the previously described complex CEP78 SV, suggestive of a recurrent genomic rearrangement. A founder haplotype was demonstrated for the latter SV in cases of Belgian and British origin, respectively. The novel 235-kb deletion was delineated using whole-genome LRS. Breakpoint analysis showed microhomology and pointed to a replication-based underlying mechanism. Moreover, data mining of bulk and single-cell human and mouse transcriptional datasets, together with CEP78 immunostaining on human retina, linked the CEP78 expression domain with its phenotypic manifestations. Overall, this study supports that the CEP78 locus is prone to distinct SVs and that SV analysis should be considered in a genetic workup of CRDHL. Finally, it demonstrated the power of sWGS and both targeted and whole-genome LRS in identifying and characterizing complex SVs in patients with ocular diseases.

14. Prenatal diagnosis of Acrofacial Dysostosis type 1 (Nager syndrome) by chromosomal microarray at the exon level

Nager syndrome or Acrofacial Dysostosis type 1 (OMIM #154400) is a rare, autosomal dominant syndrome characterized by craniofacial malformations. Fewer than 100 cases have been reported in the literature and of these, only 11 were suspected prenatally. Major phenotypic findings include micrognathia, ear anomalies and limb malformations; however, the severity is variable. Haploinsufficiency of the SF3B4 (1q21.2) gene is the most commonly recognized etiology of Nager syndrome and can be identified in more than 50% of clinically affected patients. SF3B4 encodes for the SAP49 protein which is a component of the spliceosome and has a role in the bone morphogenic protein pathway. The majority of Nager syndrome cases involve missense and nonsense mutations, or whole gene deletions. We report on a 20 year old primigravida who underwent amniocentesis at 23+1 weeks due to ultrasound findings of severe micrognathia, ear anomalies, mild hypotelorism, and absent left thumb. Chromosome microarray identified a 166kb partial deletion of the SF3B4 gene spanning exons 3-6. Copy number PCR was performed to confirm the finding and to determine whether the deletion was maternally inherited. Both independent DNA extractions of the amniotic fluid sample had a copy number of one and the maternal sample had a copy number of two, confirming the deletion. To the best of our knowledge, this prenatal diagnosis of Nager syndrome by chromosomal microarray is the first partial gene deletion of SF3B4 reported. Further, this demonstrates the importance of chromosomal microarray in correlating fetal ultrasound findings with genetic etiology. Knowledge of this result prior to birth allowed more robust family counseling about the findings and prognosis, along with adequate medical preparation for the anticipated complex interventional needs of the neonate at and after delivery.

A new case of intragenic deletion of the TCF4 gene without features of Pitt-Hopkins syndrome

Different genomic alterations affecting the TCF4 gene are usually associated with Pitt-Hopkins syndrome (PTHS). This syndrome is a rare neurodevelopmental genetic disorder characterized by distinctive facial features, abnormal breathing, psychomotor delay and severe intellectual disability (ID). The genomic alterations include whole or partial gene deletion; balanced translocation disrupting the coding sequence of the gene; and intragenic variants. The TCF4 gene encodes a basic helix-loop-helix (bHLH) transcription factor 4. Using alternative promoters, TCF4 can be transcribed from a number of alternative initial exons, allowing for translation of variable protein isoforms containing different functional domains. Full-length TCF4 has two activation domains (AD1 and AD2) that are thought to modulate transcriptional activity, a NLS domain (nuclear localization signal) that controls subcellular localization and bHLH domain. Typical PTHS patients have aberration localized between exons 9 and 18 of the gene. On the other hand, variants affecting the first protein coding exons give rise to mild non-syndromic ID. We present a ten-year-old girl with psychomotor delay and mild ID without the typical features of PTHS. Genetic investigation using array-based comparative genomic hybridization, revealed a 73.45 kb deletion within the TCF4 gene. The deletion encompassing only exon 6 (NM_001083962). This deletion was not detected in both parents. Cytogenetic analysis excluded balanced translocation disrupting the coding sequence of the gene. To the best of our knowledge, this is the first case described in literature involving only exon 6. The findings in our patients support the notion that position of the alteration in TCF4 is relevant to the phenotype. Reporting our case we want to contribute to the phenotype-genotype correlation in patients with intragenomic deletion of TCF4 gene.

Detection of &lt;i&gt;SHOX&lt;/i&gt; Gene Variations in Patients with Skeletal Abnormalities with or without Short Stature

Objective:SHOX gene mutations constitute one of the genetic causes of short stature. The clinical phenotype includes variable degrees of growth impairment, such as Langer mesomelic dysplasia (LMD), Léri-Weill dyschondrosteosis (LWD) or idiopathic short stature (ISS). The aim of this study was to describe the clinical features and molecular results of SHOX deficiency in a group of Turkish patients who had skeletal findings with and without short stature.Methods:Forty-six patients with ISS, disproportionate short stature or skeletal findings without short stature from 35 different families were included. SHOX gene analysis was performed using Sanger sequencing and multiplex ligation-dependent probe amplification analysis.Results:Three different point mutations (two nonsense, one frameshift) and one whole SHOX gene deletion were detected in 15 patients from four different families. While 4/15 patients had LMD, the remaining patients had clinical features compatible with LWD. Madelung’s deformity, cubitus valgus, muscular hypertrophy and short forearm were the most common phenotypic features, as well as short stature. Additionally, hearing loss was detected in two patients with LMD.Conclusion:This study has presented the clinical spectrum and molecular findings of 15 patients with SHOX gene mutations or deletions. SHOX deficiency should be especially considered in patients who have disproportionate short stature or forearm anomalies with or without short stature. Although most of the patients had partial or whole gene deletions, SHOX gene sequencing should be performed in suspected cases. Furthermore, conductive hearing loss may rarely accompany these clinical manifestations.