Pathogenic Frameshift Mutation Research Articles

A case report on genetic variants in CHD8, KMT2C, EP300, and TCF4 associated with autism spectrum disorder

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition influenced by several environmental and genetic factors. We present a case of a 7-year-old male with ASD, neurogenic voiding dysfunction (NVD), speech and developmental delays, and hyperactivity, and current treatment includes speech, occupational, and behavioral therapy. Family history includes consanguinity and maternal intellectual disability. Whole-exome sequencing (WES) identified maternally inherited variants in CHD8 (benign), KMT2C (likely pathogenic), and EP300 and TCF4 (uncertain significance). Despite the benign classification of the CHD8 variant, its association with ASD highlights the complexity of genotype-phenotype correlations. The likely pathogenic KMT2C frameshift mutation and deletions in EP300 and TCF4 suggest a multifactorial genetic basis for ASD in this patient. These findings highlight the importance of integrating clinical and genetic data for accurate diagnosis and personalized treatment. Whole Exome Sequencing (WES) analysis revealed these variants in the child's mother, uncle, and maternal grandfather, with the maternal uncle unaffected by ASD, ID, or ADHD, indicating potential variant interplay in disease manifestation. This case emphasizes the need for further research to elucidate the combined effects of these variants, enhancing our understanding of ASD's genetic landscape and improving clinical outcomes.

Widening the clinical, radiological and genetic spectrum of autosomal recessive ataxia of Charlevoix-Saguenay in Indian patients.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), classically presenting as a triad of early-onset cerebellar ataxia, lower extremity spasticity and peripheral neuropathy, is caused by mutations in SACS gene which encodes the protein sacsin. To provide new insight into the occurrence of SACS mutations in South India. Patients with three cardinal features of ARSACS-peripheral neuropathy, cerebellar ataxia, and pyramidal tract signs were included. Nine patients were clinically identified and genetically evaluated. Mutation screening of SACS by targeted sequencing of 40 recessive ataxia genes panel by next-generation sequencing was conducted. Additional investigations included magnetic resonance imaging (MRI), fundoscopy, optical coherence tomography (OCT) and nerve conduction studies (NCS). Functional disability was assessed by the Spinocerebellar Degeneration Functional Score. Two hundred and fifteen cerebellar ataxia patients were screened, and 9 patients with cerebellar ataxia with spasticity, peripheral neuropathy and MRI brain characteristics, consistent with a clinical diagnosis of ARSACS were identified, of which 7 patients were identified to have mutation in the SACS gene and are detailed hereafter. Age of presentation ranged from 20 to 55years (29.8 ± 11.9) with a mean disease duration of 12.7years (SD-7.65, range 5-22years). All except one had onset of symptoms in the form of an ataxic gait noticed before 20years of age. Additional features were subnormal intelligence (4/7), slow and hypometric saccades (1/7), seizures (1/7), kyphoscoliosis (1/7) and dysmorphic facies (1/7). SDFS was 3 in 5/7 patients signifying moderate disability with independent ambulation. MRI showed cerebellar atrophy with predominant atrophy of the superior vermis (7/7), horizontal linear T2 hypointensities in the pons(7/7), hyperintensities where lateral pons merges with the middle cerebellar peduncle (MCP) (7/7) well seen in fluid-attenuated inversion recovery (FLAIR) images, thickening of MCP (3/7), symmetric lateral thalamic hyperintensities (6/7), posterior fossa arachnoid cyst (4/7),thinning of posterior mid-body of corpus callosum (7/7), marginal mineralisation of the basal ganglia (7/7), bilateral parietal atrophy (7/7) and thinning of corticospinal tract on diffusion tensor imaging (DTI) (7/7). We identified pathogenic homozygous frameshift mutations in the SACS gene in six patients (including two siblings), while one patient had a heterozygous pathogenic deletion. This is the largest series of genetically confirmed ARSACS patients from India highlighting the clinical, ophthalmological, imaging and genetic features of this cohort.

Whole-exome sequencing reveals a novel frameshift mutation in a consanguineous family with a hereditary coagulation factor XII deficiency

Background and aimsWe aimed to elucidate a hereditary mutation of coagulation factor XII (FXII) in a consanguineous Chinese family. MethodsMutations were investigated using Sanger and whole-exome sequencing. FXII (FXII:C) activity and FXII antigen (FXII:Ag) were assessed using clotting assays and ELISA, respectively. Gene variants were annotated and the likelihood that amino acid mutations would affect protein function was predicted using bioinformatics. ResultsActivated partial thromboplastin time was prolonged to > 170 s (reference range, 22.3–32.5 s), and FXII:C and FXII:Ag were decreased to 0.3% and 1%, respectively, (normal range for both, 72%–150%) in the proband. Sequencing revealed a homozygous frameshift mutation c.150delC (p.Phe51Serfs*44) site in the F12 gene exon 3. This mutation results in premature termination of the encoded protein translation and the protein is truncated. Bioinformatic findings indicated a novel pathogenic frameshift mutation. ConclusionThe c.150delC frameshift mutation p.Phe51Serfs*44 in the F12 gene likely explains the low FXII level and the molecular pathogenesis of an inherited FXII deficiency in a consanguineous family.

Frameshift mutation in SQSTM1 causes proximal myopathy with rimmed vacuoles: A case report.

p62/Sequestosome-1 (SQSTM1) is a stress-inducible scaffold protein involved in multiple cellular processes, including apoptosis, inflammation, cell survival, and selective autophagy. SQSTM1 mutations are associated with a spectrum of multisystem proteinopathy, including Paget disease of the bone, amyotrophic lateral sclerosis, frontotemporal dementia, and distal myopathy with rimmed vacuoles (MRV). Herein, we report a new phenotype of SQSTM1-associated proteinopathy, a novel frameshift mutation in SQSTM1 causing proximal MRV. A 44-year-old Chinese patient presented with progressive limb-girdle weakness. She had asymmetric proximal limb weakness and myopathic features on electromyography. The magnetic resonance images showed fatty infiltration into muscles, predominantly in the thighs and medial gastrocnemius, sparing the tibialis anterior. Muscle histopathology revealed abnormal protein deposition, p62/SQSTM1-positive inclusions, and rimmed vacuoles. Next-generation sequencing showed a novel pathogenic SQSTM1 frameshift mutation, c.542_549delACAGCCGC (p. H181Lfs*66). We expanded the pathogenic genotype of SQSTM1 to include a new, related phenotype: proximal MRV. We suggest that SQSTM1 variations should be screened in cases of proximal MRV.

MPV17 mutation-related mitochondrial DNA depletion syndrome: A case series in infants.

MPV17 is a mitochondrial inner membrane protein, involved in transporting deoxynucleotides into the mitochondria. Pathogenic MPV17 mutations can cause mitochondrial deoxyribonucleic acid (DNA) depletion syndrome, which has a varied presentation with neurological, muscular and hepatic involvement. Presentation as liver failure is relatively uncommon. Here, we report four infants from four separate families with pathogenic, homozygous MPV17 mutations. All had predominant hepatic involvement with cholestasis, lactic acidosis and hypoketotic hypoglycemia. Three of them had presented with liver failure. Interestingly, one of them showed fluctuating liver functions, which worsened with infection and improved after aggressive treatment with antibiotics and supplements. Two of the four cases died in infancy, while the other two improved on conservative management with medium-chain triglyceride-based diet, vitamin supplements, co-enzyme Q and carnitine. The two surviving children are alive at 12 and 25 months of age with native liver with normal to mildly deranged liver function and no neurological dysfunction. Next-generation sequencing confirmed the diagnosis in all of our cases. One of the detected mutations, c.55delC (p.Gln19ArgfsTer3) is a novel pathogenic frameshift mutation, while another mutation c.388G>C (p.Ala130Pro), which was previously reported in Single Nucleotide Polymorphism Database in heterozygous form, is being predicted as likely pathogenic in our case series. We, therefore, propose mutation testing for MPV17 gene during evaluation of indeterminate infantile liver failure, especially those with hypoglycemia and raised plasma lactate.

Abstract B033: LP-184, a tumor site activated small molecule therapeutic, is synthetically lethal in pancreatic cancers with DNA damage repair defects

Abstract Biomarker-based therapeutic strategies associated with increased efficacy, manageable toxicity and extended disease-free survival are urgently needed in pancreatic cancer (PaCa). 15-20% of PaCa carry mutations in DNA repair pathways (BRCA1/BRCA2/PALB2/RAD51/ATR/ATM/FANCD2). Additionally, mutations in nucleotide excision repair (NER) genes (ERCC2/3/4/5/6) have been reported in ~5% of PaCa. LP-184 is an acylfulvene analog that is converted to an active alkylating agent in the strict dependency on the oxidoreductase, prostaglandin reductase 1 (PTGR1). LP-184 induced tumor cell kill is enhanced in the presence of DNA damage repair (DDR) defects. Mutation or expression driven TC-NER and/or HR deficiency is expected to predispose PaCa cells to increased sensitivity to LP-184. To test the idea of LP-184 activity in DNA repair-deficient tumors, we evaluated the anti-tumor effects of LP-184 in mouse subcutaneous PDX models harboring pathogenic frameshift mutations in ATR (CTG-1522 model) or BRCA1 (CTG-1643 model) representing HR deficiency in PaCa. Two cycles of LP-184 treatment in CTG-1522 resulted in 140% tumor growth inhibition (TGI), relative to 80% TGI with gemcitabine in this FOLFIRONOX non-responder. Similarly, complete and durable tumor regression with 112% TGI was observed in CTG-1643 with single agent LP-184. There was <5% body weight loss and no significant hematological toxicity in both these models during or after LP-184 treatment. In selected PaCa models, we determined LP-184 efficacy to be synergistic in combination with multiple treatment modalities including standard of care agent gemcitabine, and ERCC3 degrader spironolactone in vitro, and with radiation therapy (RT) in vivo. Overall Bliss synergy scores (score > 10 indicates synergy) of 12.37 for LP-184 + gemcitabine in Capan-1, and 14.08, 16.47 and 15.52 for LP-184 + spironolactone in Capan-1 (BRCA2 loss), Hs766t (ATR mutant) and Panc03.27 cell lines respectively were achieved. Taking advantage of the transcriptional regulation underlying the NRF2 mediated anti-oxidant response pathway, we reasoned that induction of PTGR1 expression, a downstream NRF2 target gene, in irradiated PaCa tumors would thereafter poise them for LP-184 treatment. Indeed, we found that Panc03.27 xenograft tumors treated with 3 mg/kg i.p. LP-184 + 4 Gy RT once weekly for 3 weeks were significantly smaller than those receiving LP-184 alone or RT alone, thus capitalizing on the opportunity to increase PTGR1 expression (and linked LP-184 anti-tumor cytotoxicity) selectively in irradiated tumors. Increased PTGR1 expression has emerged as a promising biomarker for LP-184 cytotoxicity. Moreover, LP-184 can become synthetically lethal in multiple contexts when combined with genetic or pharmacologic DDR pathway aberrations, or with RT. Based on these properties, we anticipate that LP-184 will extend therapeutic opportunities to a large subset of PaCa patients. Citation Format: Aditya Kulkarni, Diana Restifo, Troy Dos Santos, Dusica Cvetkovic, Joseph McDermott, Kishor Bhatia, Panna Sharma, Charlie Ma, Igor Astsaturov. LP-184, a tumor site activated small molecule therapeutic, is synthetically lethal in pancreatic cancers with DNA damage repair defects [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B033.

COL17A1 editing via homology-directed repair in junctional epidermolysis bullosa.

BackgroundEpidermolysis bullosa (EB), a severe genetic disorder characterized by blister formation in skin, is caused by mutations in genes encoding dermal-epidermal junction proteins that function to hold the skin layers together. CRISPR/Cas9-induced homology-directed repair (HDR) represents a promising tool for editing causal mutations in COL17A1 in the treatment of junctional epidermolysis bullosa (JEB).MethodsIn this study, we treated primary type XVII collagen (C17)-deficient JEB keratinocytes with either Cas9 nuclease or nickase (Cas9n) ribonucleoproteins (RNP) and a single-stranded oligonucleotide (ssODN) HDR template in order to correct a causal pathogenic frameshift mutation within the COL17A1 gene.ResultsAs analyzed by next-generation sequencing of RNP-nucleofected keratinocytes, we observed an HDR efficiency of ∼38% when cells were treated with the high-fidelity Cas9 nuclease, a mutation-specific sgRNA, and an ssODN template. The combined induction of end-joining repair and HDR-mediated pathways resulted in a C17 restoration efficiency of up to 60% as assessed by flow cytometry. Furthermore, corrected JEB keratinocytes showed a significantly increased adhesive strength to laminin-332 and an accurate deposition of C17 along the basement membrane zone (BMZ) upon differentiation into skin equivalents.ConclusionHere we present a gene editing approach capable of reducing end joining-generated repair products while increasing the level of seamless HDR-mediated gene repair outcomes, thereby providing a promising CRISPR/Cas9-based gene editing approach for JEB.

MEDB-57.GNAS inactivation as a driver for sonic hedgehog-activated medulloblastoma

INTRODUCTION: Sonic hedgehog (SHH)-activated medulloblastoma is one of the four consensus molecular subgroups of medulloblastoma and is typically associated with PTCH1, SUFU, and/or SMO mutations. GNAS inactivating mutations are a less commonly recognized tumorigenic driver for SHH pathway activation. CASE PRESENTATION: We report the case of an 11-month-old male who presented with a large posterior fossa mass, cerebellar tonsillar herniation, and obstructive hydrocephalus. Following a successful gross total resection, pathology was consistent with desmoplastic/nodular medulloblastoma. Tumor molecular profiling using a DNA-based, next-generation sequencing platform detected a pathogenic frameshift mutation in the GNAS gene (p.D189Mfs*14, NM_000516.4:c.565_568del) at a variant allele frequency of 81%, suggestive of biallelic GNAS inactivation. The same mutation was detected from a buccal swab sample, confirming germline GNAS inactivation. Whole genome methylation profiling was consistent with medulloblastoma subclass SHH B (infant). Concurrent with this brain tumor evaluation, a skin biopsy was performed of scattered subcutaneous, plate-like nodules distributed over the patient’s back and extremities. This revealed metaplastic-appearing cancellous bone within the dermis and subcutaneous tissue consistent with plate-like osteoma cutis. He was treated with high-dose chemotherapy followed by autologous stem cell rescue and remains disease-free 15 months from diagnosis. DISCUSSION: There have been scattered case reports describing germline loss-of-function GNAS mutations acting as tumorigenic drivers of SHH medulloblastoma. However, GNAS alterations are not covered by most standard diagnostic molecular sequencing panels for medulloblastoma. Other phenotypic manifestations of germline inactivating GNAS mutations include pseudohypoparathyroidism, pseudopseudohypoparathyroidism, progressive osseous heteroplasia, and osteoma cutis. CONCLUSION: Knowledge of the possible association between germline GNAS inactivating mutations and the development of childhood SHH-activated medulloblastoma is essential for prompt diagnosis and treatment initiation. As such, consideration should be given for inclusion of GNAS alterations in diagnostic medulloblastoma sequencing panels, especially in the setting of osteoma cutis or other endocrinopathies.

239 New de novo mutation in CACNA1a gene leading to episodic ataxia type 2

Introduction andAimEpisodic ataxia (EA) is a group of inherited ion channel disorders affecting the cer- ebellum characterised by sporadic episodes of ataxia (Riant et al, 2011; Tomlinson et al, 2013). At least 7 different types have been identified, type 2 being the most common. We describe a patient present- ing with recurrent episodes of dizziness and gait disturbance with a novel mutation in CACNA1a gene (c.586del) leading to episodic ataxia type 2.Clinical presentationA 65-year old male presented with recurrent episodes of dizziness and gait distur- bance, which had been present since childhood but increased in severity over last 2 years and leading to hospitalisation. He also reported mild cognitive impairment. Examination showed bilateral horizontal jerky nyastagmus with an otherwise normal neurological examination.InvestigationsNeuroimaging studies showed mild small vessel disease. Genetic sequencing revealed a heterogenicity for the c.586del likely pathogenic frameshift mutation in exon 4 of the CACNA1A gene, resulting in targeting of the mRNA for nonsense- mediated decay or the production of a truncated protein leading to the diagnosis of autosomal dominant episodic ataxic type 2 in this patient.SummaryWe report a new de novo mutation in CACNA1a gene leading to episodic ataxia type 2. The patient was given acetazolamide (250mg/day) which improved his symptoms. Episodic ataxia should be considered in patients presenting with a history of intermittent dizziness.kitwu@doctors.org.uk

Purification of the human cone‐rod homeobox protein

A “lock and key” is the most common depiction of protein binding, protein has a specific structure, and its binding molecule of interest can fit into it. What happens when a protein domain needs to bind to multiple biomolecules? The transcription factor CRX is one of those with an activation domain that can bind to various biomolecules. CRX regulates the gene expression of cone and rod photoreceptors. To effectively regulate gene expression, CRX’s activation domain interacts with multiple transcriptional co‐activators. Unlike the “lock and key” concept with defined structures, we propose that the activation domain of CRX is intrinsically disordered, allowing for interactions of multiple proteins in the same domain. In support of a disordered activation domain, we observe that proline and serine amino acids are abundant in the CRX activation domain. We also note an abundance of pathogenic frameshift mutations and a lack of point mutants in the activation domain. To characterize the structure of CRX, we attempted to purify full‐length CRX and CRX coexpressed with known transcriptional coactivator NRL. We hypothesize that this latter experiment will enhance the stability and expression of full‐length CRX. We have purified CRX using nickel chromatography and are currently optimizing the next steps to enhance the purity of CRX and NRL. The information from biochemical and structural experiments on purified CRX will provide insight into the transcriptional regulation of CRX, leading to the development of photoreceptor neurons.

A novel TRMT5 mutation causes a complex inherited neuropathy syndrome: The role of nerve pathology in defining a demyelinating neuropathy.

We aim to present data obtained from three patients belonging to three unrelated families with an infantile onset demyelinating neuropathy associated to somatic and neurodevelopmental delay and to describe the underlying genetic changes. We performed whole-exome sequencing on genomic DNA from the patients and their parents and reviewed the clinical, muscle and nerve data, the serial neurophysiological studies, brain and muscle MRIs, as well as the respiratory chain complex activity in the muscle of the three index patients. Computer modelling was used to characterise the new missense variant detected. All three patients had a short stature, delayed motor milestone acquisition, intellectual disability and cerebellar abnormalities associated with a severe demyelinating neuropathy, with distinct morphological features. Despite the proliferation of giant mitochondria, the mitochondrial respiratory chain complex activity in skeletal muscle was normal, except in one patient in whom there was a mild decrease in complex I enzyme activity. All three patients carried the same two compound heterozygous variants of the TRMT5 (tRNA Methyltransferase 5) gene, one known pathogenic frameshift mutation [c.312_315del (p.Ile105Serfs*4)] and a second rare missense change [c.665 T > C (p.Ile222Thr)]. TRMT5 is a nuclear-encoded protein involved in the post-transcriptional maturation of mitochondrial tRNA. Computer modelling of the human TRMT5 protein structure suggests that the rare p.Ile222Thr mutation could affect the stability of tRNA binding. Our study expands the phenotype of mitochondrial disorders caused by TRTM5 mutations and defines a new form of recessive demyelinating peripheral neuropathy.

Clinicopathological and molecular characterization of biphasic hyalinizing psammomatous renal cell carcinoma: further support for the newly proposed entity

The classification of renal neoplasms continues to evolve with novel, emerging, and provisional entities being described constantly. Biphasic hyalinizing psammomatous renal cell carcinoma (BHP RCC) associated with somatic NF2 mutations is one such new renal entity and is considered as a provisional category of RCC due to its very limited data. To provide further support for the newly proposed entity, we identified three additional cases of BHP RCC, with clinicopathological, immunohistochemical, and various molecular analyses. There were 2 males and 1 female, aged 65, 56, and 69 years, respectively. The neoplasms were unencapsulated, and all had a characteristic biphasic appearance of smaller cells clustering around basement membrane material within larger acini, forming pseudorosettes or a glomeruloid pattern. Hyalinized sclerotic stroma and psammoma bodies were abundant in two cases and focally present in one case. Focal areas of a less distinctive appearance were also noted; one additionally had an elongated tubular pattern in the myxoid stroma that is reminiscent of mucinous tubular and spindle cell carcinoma; one consisted solid alveolar architectures of epithelioid clear cells, bearing some resemblance to clear cell RCC. The neoplasms did not have a distinctive immunohistochemistry (IHC) profile, though all labeled for vimentin and CK7. Targeted DNA sequencing revealed that one case harbored a pathogenic somatic frameshift mutation in the NF2 gene, which was further confirmed by Sanger sequencing. The other two cases lacked NF2 mutations and instead demonstrated NF2 promoter methylation by methylation-specific polymerase chain reaction. Subsequent IHC assessment showed loss of expression of NF2 in all 3 cases, which evaluated NF2 status at the protein level. According to RNA sequencing-based clustering analysis, the 3 cases formed a distinct group with a shared specific transcriptional profile different from that of other established renal tumor types. In addition, phosphate inositide 3-kinase (PI3K)/AKT pathway was enriched significantly and on the top of all enriched pathways. Clinically, one patient developed bone metastases and died of disease two years after diagnosis. The other two patients had no evidence of recurrence or metastases, at 4- and 5-yearfollow-up. These findings not only validate previously described clinicopathological featuresbut also expand the potentially genetic alterations and available clinical outcome data.

A Rare Pathogenic Frameshift Mutation in NSD1 Gene Related to Sotos Syndrome in a Turkish Patient

A Rare Pathogenic Frameshift Mutation in NSD1 Gene Related to Sotos Syndrome in a Turkish Patient

Analyses Mutations in GSN, CST3, TTR, and ITM2B Genes in Chinese Patients With Alzheimer's Disease.

Amyloid protein deposition is a common mechanism of hereditary amyloidosis (HA) and Alzheimer’s disease (AD). Mutations of gelsolin (GSN), cystatin C (CST3), transthyretin (TTR), and integral membrane protein 2B (ITM2B) genes can lead to HA. But the relationship is unclear between these genes and AD. Genes targeted sequencing (GTS), including GSN, CST3, TTR, and ITM2B, was performed in a total of 636 patients with clinical AD and 365 normal controls from China. As a result, according to American College of Medical Genetics and Genomics (ACMG) guidelines, two novel likely pathogenic frame-shift mutations (GSN:c.1036delA:p.K346fs and GSN:c.8_35del:p.P3fs) were detected in five patients with AD, whose initial symptom was memory decline, accompanied with psychological and behavioral abnormalities later. Interestingly, the patient with K346fs mutation, presented cerebral β-amyloid protein deposition, had an early onset (48 years) and experienced rapid progression, while the other four patients with P3fs mutation had a late onset [(Mean ± SD): 69.50 ± 5.20 years] and a long course of illness [(Mean ± SD): 9.24 ± 4.86 years]. Besides, we also discovered 17 variants of uncertain significance (VUS) in these four genes. To our knowledge, we are the first to report AD phenotype with GSN mutations in patients with AD in the Chinese cohort. Although mutations in the GSN gene are rare, it may explain a small portion of clinically diagnosed AD.

LINS1-associated neurodevelopmental disorder: Family with novel mutation expands the phenotypic spectrum.

ObjectiveClinical, neuroimaging, and genetic characterization of 3 patients with LINS1-associated developmental regression, intellectual disability, dysmorphism, and further neurologic deficits.MethodsThree affected brothers from a consanguineous family from Afghanistan, their 2 healthy siblings, and both parents were all assessed in the clinic. General and neurologic examination, expert dysmorphology examination, and 3T brain MRI were performed. Whole-exome sequencing was performed for the 3 affected brothers, followed by Sanger sequencing in all available family members.ResultsThe index patient and his 2 affected brothers presented a complex neurologic syndrome with similar features but marked intrafamilial phenotypical variability, including varying degrees of cognitive impairment, speech impairment, dystonia, abnormal eye movements, and dysmorphic features. All 3 affected brothers are homozygous for a novel, pathogenic frameshift mutation in LINS1, c.1672_1679del, and p.Gly558Profs*22, whereas both parents and healthy siblings are heterozygous for the mutation. No major brain malformations were evident in 3T brain MRI of the affected brothers.ConclusionThis consanguineous family with a novel mutation expands the spectrum of LINS1-associated disorder to include developmental regression, oculomotor signs, and dystonia, previously not described in the published 9 cases of this rare disorder. The 3T-MRI data from our 3 patients and review of the neuroimaging data in the literature showed unspecific brain MRI changes. LINS1 protein is a known modulating factor of the Wnt signaling pathway, with important roles in organogenesis including of the cerebral cortex. More research is warranted to disentangle the underlying pathophysiologic mechanisms, leading to cognitive impairment and the complex phenotype of LINS1-associated disorder.

A case of Turcot\u2019s syndrome type 1 with loss of immunoexpression of MSH6 in colon cancer and liver metastasis due to secondary somatic mutation in coding mononucleotide (C)8 tract: a case report

BackgroundLynch syndrome (LS), which is known as a hereditary cancer syndrome, is distinguished by microsatellite instability, represented by the altered number of repetitive sequences in the coding and/or non-coding region. Immunohistochemical staining (IHC) of DNA mismatch repair (MMR) proteins (e.g., MLH1, MSH2, MSH6, and PMS2) has been recognized as an useful technique for screening of LS. Previous study has shown that the assessment of IHC, however, requires specific caution due to variable staining patterns even without germline mutations in MMR genes.Case presentationA 48-year-old man, who had been treated for anaplastic astrocytoma, was referred to our department for the precise examination of progressing anemia. Whole-body examination revealed two advanced carcinomas in descending colon and stomach. A hypo-vascular mass lesion was detected in liver as well. Pathological diagnosis (on surgical specimens) was poorly differentiated adenocarcinoma in descending colon, moderately differentiated tubular adenocarcinoma in stomach, and liver metastasis, which is possibly from colon. It was suspected that this case would be Turcot’s syndrome-type-1 due to its specific family history having two cases of colon cancer within the second relatives. Pathogenic frameshift mutations in codon 618 of MLH1 gene was identified. Immunohistochemical analyses (IHC) demonstrated complete loss of MLH1 immuno-expression as well as of PMS2 except for those in brain tumor. Although frameshift mutation was not found in MSH6 gene, histological expression of MSH6 was patchy in primary colon carcinoma and was completely lost in the metastatic site in liver. MSH6 expression in gastric carcinoma, a coincidental cancer in this case, was intact. An abnormal (C)8 region was identified by the cloned PCR of colon and liver tumors but not from gastric cancer. Frameshift mutation in a (C)8 tract in exon 5 of the MSH6 gene was also detected in liver metastasis.ConclusionThis case supports a plausible mechanism, proposed by a previous literature, for the reduced expression of MSH6 in a somatic mutation manner, which might preferentially happen in colon cancer rather than in stomach carcinoma in MLH1/PMS2-deficient type of Turcot’s syndrome type 1.

Identification of two novel mutations in POU4F3 gene associated with autosomal dominant hearing loss in Chinese families.

Autosomal dominant non‐syndromic hearing loss is genetically heterogeneous with 47 genes identified to date, including POU4F3. In this study, by using a next‐generation sequencing panel targeting 127 deafness genes, we identified a pathogenic frameshift mutation c.704_705del and a missense mutation c.593G>A in two three‐generation Chinese families with late‐onset progressive ADNSHL, respectively. The novel mutations of POU4F3 co‐segregated with the deafness phenotype in these two families. c.704_705del caused a frameshift p.T235fs and c.593G>A caused an amino acid substitution of p.R198H. Both mutations led to an abnormal and incomplete protein structure. POU4F3 with either of the two mutations was transiently transfected into HEI‐OC1 and HEK 293 cell lines and immunofluorescence assay was performed to investigate the subcellular localization of mutated protein. The results indicated that both c.704_705del (p.T235fs) and c.593G>A (p.R198H) could impair the nuclear localization function of POU4F3. The p.R198H POU4F3 protein was detected as a weak band of the correct molecular weight, indicating that the stability of p.R198H POU4F3 differed from that of the wild‐type protein. While, the p.T235fs POU4F3 protein was expressed with a smaller molecular weight, implying this mutation result in a frameshift and premature termination of the POU4F3 protein. In summary, we report two novel mutations of POU4F3 associated with progressive ADNSHL and explored their effects on POU4F3 nuclear localization. These findings expanded the mutation spectrum of POU4F3 and provided new knowledge for the pathogenesis of POU4F3 in hearing loss.

SAT-LB310 A Mysterious Multiple Endocrine Neoplasia (MEN) Like Syndrome

Multiple endocrine neoplasia (MEN) is characterized by the occurrence of tumors involving two or more endocrine glands in a single patient. Among the four MEN syndromes, MEN4 due to CDKN1B mutation is characterized by parathyroid and anterior pituitary tumors in possible association with tumors of the adrenals, kidneys, and reproductive organs. We presented a patient with MEN 4 like syndrome without CDKN1B, menin or RET mutations.74 year old male was diagnosed with acromegaly and primary hyperparathyroidism at age 63. Genetic testing revealed no mutations in menin and RET genes. At age 68, he was diagnosed with renal cell carcinoma (RCC) and at age 70, 2cm left adrenal mass was identified on surveillance computerized tomography (CT). No biochemical workup was pursued. Four years later, he developed hypertensive crisis during spine surgery at our institution. Workup revealed elevated plasma metanephrine (490 pg/ml, normal <57) and normetanephrine (1333 pg/ml, normal <148). CT showed the left adrenal mass increased in size to 4.5 cm. Family history is negative for any endocrine tumors. He underwent repeat genetic testing. Analyses of 133 gene panel reported no germline mutations in menin, RET, CDKN1B, NF12, VHL, SDH and other genes tested but there were variants of uncertain significance (VUS) identified in CHEK2 c.14C>T (p.Ser5Leu) and PTCH2 c.2812G>A (p.Gly938Ser). Patient successfully underwent left adrenalectomy after alpha blockage. Paired tumor-normal sequencing of the resected tumor detected a pathogenic deletion frameshift mutation in NF1 with loss of heterozygosity (LOH) along with copy number alterations with losses in 1p34.1-p11.2, 11p11.2-15.4, 11q14.1-q25 and 17q11.2 (including NF1). VUSs were also detected including CDKN1A C117Y variant, and CHD2P80L. Since germline and tumor testing failed to reveal any known pathogenic variants, whole exome sequencing (pending) will be pursued.The presentation with RCC, pheochromocytoma, pituitary adenoma and parathyroid adenoma is consistent with a MEN syndrome in this patient despite no known pathogenic MEN mutations detected. Somatic mutation in NF1 is a common finding in pheochromocytoma. The biochemical phenotype of pheochromocytoma (elevated metanephrines) is consistent with cluster 2 tumors of kinase signaling pathway as seen in tumors of MEN syndrome and neurofibromatosis. We hope to gain more insight via whole exome sequencing to evaluate for potential novel gene mutation(s).

A novel pathogenic variant in OSBPL2 linked to hereditary late-onset deafness in a Mongolian family

BackgroundTo investigate the clinical features and the underlying causal gene of a family with hereditary late-onset deafness in Inner Mongolia of China, and to provide evidence for the early genetic screening and diagnosis of this disease.MethodsFamily data were collected to draw a pedigree. Audiological testing and physical examination of the family members were conducted following questionnaire. Genomic DNA was extracted from peripheral blood of 5 family members (3 patients and 2 normal control) and subjected to whole genome sequencing for identifying deafness casual genes. The pathogenic variant in the deafness gene was further confirmed by Sanger sequencing.ResultsThe family is composed of a total of 6 generations, with 53 traceable individuals. In this family,19 of them were diagnosed with post lingual deafness with the age of onset between 10 and 40 years, displaying delayed and progressive hearing loss. Patients with hearing loss showed bilateral symmetry and mild to severe sensorineural deafness. The pattern of deafness inheritance in this family is autosomal dominant. Whole genome sequencing identified a novel pathogenic frameshift mutation, c.158_159delAA (p.Gln53Arg fs*100) in the gene OSBPL2 (Oxysterol-binding protein-related protein 2, NM_144498.2), which is absent from genomic data of 201 unrelated normal subjects. This pathogenic variant was further validated by Sanger sequencing, and was found to co-segregate in this family.ConclusionsWhole genome sequencing identified a two-nucleotide deletion in OSBPL2 (c.158_159delAA) as the pathogenic variant for deafness in the family. Our finding expands the mutational spectrum of OSBPL2 and contributes to the pathogenic variant list in genetic counseling for deafness screening.

Liddle syndrome misdiagnosed as primary aldosteronism resulting from a novel frameshift mutation of SCNN1B.

Liddle syndrome (LS), a monogenetic autosomal dominant disorder, is mainly characterized by early-onset hypertension and hypokalemia. Clinically, misdiagnosis or missing diagnosis is common, since clinical phenotypes of LS are variable and nonspecific. We report a family with misdiagnosis of primary aldosteronism (PA), but identify as LS with a pathogenic frameshift mutation of the epithelial sodium channel (ENaC) β subunit. DNA samples were collected from a 32-year-old proband and 31 other relatives in the same family. A designed panel including 41 genes associated with monogenic hypertension was screened using next-generation sequencing. The best candidate disease-causing variants were verified by Sanger sequencing. Genetic analysis of the proband revealed a novel frameshift mutation c.1838delC (p.Pro613Glnfs*675) in exon 13 of SCNN1B. This heterozygous mutation involved the deletion of a cytosine from a string of three consecutive cytosines located at codons 612 to 613 and resulted in deletion of the crucial PY motif and elongation of the β-ENaC protein. The identical mutation was also found in 12 affected family members. Amiloride was effective in alleviating LS for patients. There were no SCNN1A or SCNN1G mutations in this family. Our study emphasizes the importance of considering LS in the differential diagnosis of early-onset hypertension. The identification of a novel frameshift mutation of SCNN1B enriches the genetic spectrum of LS and has allowed treatment of this affected family to prevent severe complications.