C-terminal Frameshift Mutations Research Articles

Identification of a Novel Frameshift variant of the ATRX gene: a Case Report and Review of the genotype–phenotype relationship

BackgroundX-linked intellectual disability-hypotonic facies syndrome-1 (MRXHF1) and Alpha-thalassemia X-linked intellectual disability (ATR-X) syndrome are caused by pathogenic variant in the ATRX gene, a member of the switch/sucrose non-fermentable (SWI-SNF) protein family that exhibits chromatin remodeling activity. These syndromes show a wide spectrum of clinical manifestations, such as distinctive dysmorphic features, mild-to-profound intellectual disability, motor development delay, seizures, urogenital abnormalities, and gastrointestinal disorders.Case presentation and literature reviewA 3-year-old boy from a Chinese non-consanguineous family was diagnosed with MRXHF1 by whole-exome sequencing. Comprehensive family history information was obtained. The Medline database was searched until 1st Aug 2023 for articles related to ATRX pathogenic variant. Data on gene/protein mutations and clinical symptoms were extracted. The proband showed intellectual disability, motor development delay, typical facial abnormalities, urogenital defect, behavior problems, and optical nerve dysplasia. A novel frameshift mutation c.399_400dup, (p.Leu134Cysfs*2) in the ATRX gene was the primary cause, which occurs right before the ATRXDNMT3-DNMT3L (ADD) domain of ATRX protein. Missense mutation is the most common variation type. The ADD and helicase-like domains are the most frequently affected domains. Epilepsy, congenital heart disease, urogenital defect, acoustic defect, and optical defect are more prevalent in patients with frameshift mutations compared to those with missense mutations. There are more urogenital defects with C-terminal frameshift mutations than with N-terminal frameshift mutations.ConclusionWe described a novel frameshift mutation in the ATRX gene in a patient with MRXHF1 syndrome and summarized the genotype–phenotype relationship of ATRX pathogenic variant by variation type and affected protein domain. The regulatory mechanism underlying ATRX variant requires comprehensive analysis in future studies.

RUNX1 Germline Mutations of Autosomal-Dominant Familial Platelet Disorder Are More Common Than Expected in Adult Myeloid Leukemias Indicating Highly Variable Penetrance

Germline (GL) RUNX1 (RUNX1GL) mutations result in autosomal-dominant familial platelet disorder (FPD) and are associated with heightened risk of myeloid neoplasia (MN). Indeed about 43% of RUNX1GL carriers are expected to develop MN, by the age of 50 yrs. 1 AML progression can be accompanied by biallelic RUNX1 hits. The phenotypic heterogeneity, incomplete penetrance, and leukemogenic risk depend on mutational burden and functional impact (topology of mutations, configuration) of RUNX1 and thus evolution of MN may be delayed. 2-3 We hypothesized that a large systematic study of RUNX1GL including types and topology will elucidate the impact of various RUNX1mutations ( RUNX1MT), their configuration, and molecular associations to provide prognostic insights and mechanistic conclusions as to the pathogenesis of these mutations. We analyzed over 9000 patients from Cleveland Clinic Foundation (CCF; n=7953) and public series. 4-5 We devised a novel rational algorithm to assess of RUNX1GL variants ( Fig1A). Stringent criteria were used that prioritized exclusion of false positives: GL benign and likely benign alterations were not included, while pathogenic and likely pathogenic (P/LP) variants were further curated. Carriers of VUS RUNX1MT that displayed past medical history or family history suspicious for FPD or leukemia were classified as VUS of suspicious pathogenicity (sVUS). The remaining variants, including P/LP, were further filtered and clinically annotated ( Fig1B). GL confirmation was possible in 22 alterations, e.g., wherein our algorithm resulted in ambivalent assignment. We found 627 RUNX1 variants (GL and somatic) in 557 patients, of which 8 were sVUS and 106 (17%) were found to be GL P/LP including 3 new RUNX1GL alterations (Y281*, S141L, S410*). The mean age at presentation of RUNX1GL carriers was lower (64 yrs.) compared to patients with somatic RUNX1MT(70 yrs., P= .0053). There was no difference in disease phenotype (AML, MDS, MDS/MPN) between GL, somatic, and biallelic (somatic/somatic) RUNX1MT carriers. Of note, 1 patient with predicted RUNX1GL variants had aplastic anemia. Cytogenetic analysis revealed that 68% of RUNX1GL cases were associated with normal karyotype. Four RUNX1GL cases also displayed trisomy 21. FLT3 ITD/PTDwas more prevalent in RUNX1GL carriers (14.8%) than in those with somatic RUNX1MT(4.7%) ( P= .021). PHF6 MT were more frequent in carriers of RUNX1GL (18%) compared to somatic RUNX1 carriers (9%) ( P= .078). Patients with a second RUNX1 hit included somatic biallelic (n=52)and GL/somatic (n=6) RUNX1 MT. The most common mutations co-occuring with biallelic RUNX1 hit werethose in EZH2 (27%, P= .025), ZRSR2 (19%, P= .020), U2AF1 (21%, P= .030) and PTPN11 (16%, P= .013) compared to monoallelic RUNX1MT. We further categorized RUNX1MT according to their predicted functional effects as: i) haploinsufficient (RUNT-truncating mutations); ii) dominant negative (post-RUNT nonsense, C-terminal frameshift, and missense mutations previously described as dominant negative); and iii) RUNT missense mutations. 6-7 We analyzed 93 (25%) mutations classified as haploinsufficient, 98 (27%) as dominant negative, and 134 (36%) as RUNT missense. There was no difference in the topology between somatic and GL hits. Survival analysis indicated that there was no difference in prognosis between dominant negative, haploinsufficient, and RUNT missense mutations ( P= .59). Among RUNX1GL carriers, there was also no prognostic difference between subtypes of RUNX1GL ( P= .896). To determine whether GL mutation types convey a greater penetrance, we compared the age of patients at presentation for somatic and GL. Specifically, carries of GL dominant negative mutations were diagnosed at a younger median age (60.5 yrs.) compared to carriers of GL haploinsufficient (68 yrs.) and RUNT missense (69 yrs.). However, this trend was not statistically significant. To rule out the effect of RUNX1GL lesions on genome instability, we quantified the mutational burden per each GL variant. Compared to RUNT missense, haploinsufficient GL variants were associated with a greater number of co-occurring mutations ( P= .013) while dominant negative GL variants were not ( P= .054). In summary, our study of the topological features of RUNX1GL variants showed that dominant negative RUNX1 mutations are frequent in MN and are not associated with specific phenotypes, but rather define a variable penetrance.

High clinical heterogeneity in a Chinese pedigree of retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S)

BackgroundBeing a newly defined disease, RVCL-S is underrecognized by clinicians globally. It is an autosomal dominantly inherited small vessel disease caused by the heterozygous C-terminal frameshift mutation in TREX1 gene. RVCL-S is featured by cerebral dysfunction, retinopathy, and vasculopathy in multiple internal organs. Misdiagnosis may cause devastating consequences in patients, such as iatrogenic PML caused by misuse of immunosuppressants. Thus, increasing awareness of this disease is in urgent need.ResultsWe uncovered a large Chinese origin RVCL-S pedigree bearing the TREX1 mutation. A comprehensive characterization combining clinical, genetic, and neuropathological analysis was performed. The Intrafamilial comparison showed highly heterogeneous clinical phenotypes. Mutation carriers in our pedigree presented with retinopathy (8/13), seizures (2/13), increased intracranial pressure (1/13), mild cognitive impairment (3/13), stroke-like episode (3/13), mesenteric ischemia (1/13), nephropathy (9/13), ascites (3/13), hypertension (9/13), hyperlipidemia (3/8), hypoalbuminemia (3/8), normocytic anemia (3/8), subclinical hypothyroidism (1/8), hyperfibrinogenemia (1/8), hyperparathyroidism (2/8), and abnormal inflammatory markers (4/8). The constellation of symptoms is highly varied, making RVCL-S a challenging diagnosis. Comparison with reported RVCL-S pedigrees further revealed that the mesenteric ischemia is a novel clinical finding and the MRS pattern of brain lesions is emulating neoplasm and tumefactive demyelination.ConclusionOur reports characterize a highly heterogeneous RVCL-S pedigree, highlight the probability of misdiagnosis in clinical practice, and broaden the clinical spectrum of RVCL-S.

PIK3CA C-terminal frameshift mutations are novel oncogenic events that sensitize tumors to PI3K-α inhibition

PIK3CA hotspot mutation is well established as an oncogenic driver event in cancer and its durable and efficacious inhibition is a focus in the development and testing of clinical cancer therapeutics. However, hundreds of cancer-associated PIK3CA mutations remain uncharacterized, their sensitivity to PI3K inhibitors unknown. Here, we describe a series of PIK3CA C-terminal mutations, primarily nucleotide insertions, that produce a frame-shifted protein product with an extended C terminus. We report that these mutations occur at a low frequency across multiple cancer subtypes, including breast, and are sufficient to drive oncogenic transformation in vitro and in vivo. We demonstrate that the oncogenicity of these mutant p110α proteins is dependent on p85 but not Ras association. P110α-selective pharmacologic inhibition blocks transformation in cells and mammary tumors characterized by PIK3CA C-terminal mutation. Taken together, these results suggest patients with breast and other tumors characterized by PIK3CA C-terminal frameshift mutations may derive benefit from p110α-selective inhibitors, including the recently FDA-approved alpelisib.

Increased Mortality and Vascular Phenotype in a Knock-In Mouse Model of Retinal Vasculopathy With Cerebral Leukoencephalopathy and Systemic Manifestations.

Background and Purpose- Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is an autosomal dominant small vessel disease caused by C-terminal frameshift mutations in the TREX1 gene that encodes the major mammalian 3' to 5' DNA exonuclease. RVCL-S is characterized by vasculopathy, especially in densely vascularized organs, progressive retinopathy, cerebral microvascular disease, white matter lesions, and migraine, but the underlying mechanisms are unknown. Methods- Homozygous transgenic RVCL-S knock-in mice expressing a truncated Trex1 (three prime repair exonuclease 1) protein (similar to what is seen in patients) and wild-type littermates, of various age groups, were subjected to (1) a survival analysis, (2) in vivo postocclusive reactive hyperemia and ex vivo Mulvany myograph studies to characterize the microvascular and macrovascular reactivity, and (3) experimental stroke after transient middle cerebral artery occlusion with neurological deficit assessment. Results- The mutant mice show increased mortality starting at midlife (P=0.03 with hazard ratio, 3.14 [95% CI, 1.05-9.39]). The mutants also show a vascular phenotype as evidenced by attenuated postocclusive reactive hyperemia responses (across all age groups; F[1, 65]=5.7, P=0.02) and lower acetylcholine-induced relaxations in aortae (in 20- to 24-month-old mice; RVCL-S knock-in: Emax: 37±8% versus WT: Emax: 65±6%, P=0.01). A vascular phenotype is also suggested by the increased infarct volume seen in 12- to 14-month-old mutant mice at 24 hours after infarct onset (RVCL-S knock-in: 75.4±2.7 mm3 versus WT: 52.9±5.6 mm3, P=0.01). Conclusions- Homozygous RVCL-S knock-in mice show increased mortality, signs of abnormal vascular function, and increased sensitivity to experimental stroke and can be instrumental to investigate the pathology seen in patients with RVCL-S.

Intestinal dysmotility in a zebrafish (Danio rerio) shank3a;shank3b mutant model of autism

Background and aimsAutism spectrum disorder (ASD) is currently estimated to affect more than 1% of the world population. For people with ASD, gastrointestinal (GI) distress is a commonly reported but a poorly understood co-occurring symptom. Here, we investigate the physiological basis for GI distress in ASD by studying gut function in a zebrafish model of Phelan-McDermid syndrome (PMS), a condition caused by mutations in the SHANK3 gene.MethodsTo generate a zebrafish model of PMS, we used CRISPR/Cas9 to introduce clinically related C-terminal frameshift mutations in shank3a and shank3b zebrafish paralogues (shank3abΔC). Because PMS is caused by SHANK3 haploinsufficiency, we assessed the digestive tract (DT) structure and function in zebrafish shank3abΔC+/− heterozygotes. Human SHANK3 mRNA was then used to rescue DT phenotypes in larval zebrafish.ResultsSignificantly slower rates of DT peristaltic contractions (p < 0.001) with correspondingly prolonged passage time (p < 0.004) occurred in shank3abΔC+/− mutants. Rescue injections of mRNA encoding the longest human SHANK3 isoform into shank3abΔC+/− mutants produced larvae with intestinal bulb emptying similar to wild type (WT), but still deficits in posterior intestinal motility. Serotonin-positive enteroendocrine cells (EECs) were significantly reduced in both shank3abΔC+/− and shank3abΔC−/− mutants (p < 0.05) while enteric neuron counts and overall structure of the DT epithelium, including goblet cell number, were unaffected in shank3abΔC+/− larvae.ConclusionsOur data and rescue experiments support mutations in SHANK3 as causal for GI transit and motility abnormalities. Reductions in serotonin-positive EECs and serotonin-filled ENS boutons suggest an endocrine/neural component to this dysmotility. This is the first study to date demonstrating DT dysmotility in a zebrafish single gene mutant model of ASD.

Sclerosing bone dysplasias with hallmarks of dysosteosclerosis in four patients carrying mutations in SLC29A3 and TCIRG1.

The osteopetroses and related sclerosing bone dysplasias can have a broad range of manifestations. Especially in the milder forms, sandwich vertebrae are an easily recognizable and reliable radiological hallmark. We report on four patients from three families presenting with sandwich vertebrae and platyspondyly. The long bone phenotypes were discordant with one patient showing modeling defects and patchy osteosclerosis, while the second displayed only metaphyseal sclerotic bands, and the third and fourth had extreme metaphyseal flaring with uniform osteosclerosis. Two of the four patients had experienced pathological fractures, two had developmental delay, but none showed cranial nerve damage, hepatosplenomegaly, or bone marrow failure. According to these clinical features the diagnoses ranged between intermediate autosomal recessive osteopetrosis and dysosteosclerosis. After exclusion of mutations in CLCN7 we performed gene panel and exome sequencing. Two novel mutations in SLC29A3 were found in the first two patients. In the third family a TCIRG1 C-terminal frameshift mutation in combination with a mutation at position +4 in intron 2 were detected. Our study adds two cases to the small group of individuals with SLC29A3 mutations diagnosed with dysosteosclerosis, and expands the phenotypic variability. The finding that intermediate autosomal recessive osteopetrosis due to TCIRG1 splice site mutations can also present with platyspondyly further increases the molecular heterogeneity of dysosteosclerosis-like sclerosing bone dysplasias.

Functional characterization of recurrent FOXA2 mutations seen in endometrial cancers.

FOXA2, a member of the forkhead family of DNA-binding proteins, is frequently mutated in uterine cancers. Most of the mutations observed in uterine cancers are frameshifts and stops. FOXA2 is considered to be a driver gene in uterine cancers, functioning as a haploinsufficient tumor suppressor. The functional consequences of FOXA2 mutations, however, have not yet been determined. We evaluated the effects that frameshift mutations and a recurrent missense mutation have on FOXA2 transcriptional activity. Recurrent N-terminal frameshifts resulted in truncated proteins that failed to translocate to the nucleus and have no transcriptional activity using an E-cadherin/luciferase reporter assay. Protein abundance was reduced for the recurrent p.S169 W mutation, as was transcriptional activity. A C-terminal frameshift mutation had increased FOXA2 levels evidenced by both Western blot and immunofluorescence. Given that FOXA2 is a recognized activator of E-cadherin (CDH1) expression and E-cadherin's potential role in epithelial-to-mesenchymal transition in a wide range of cancer types, we tested the hypothesis that FOXA2 mutations in primary uterine cancer specimens would be associated with reduced CDH1 transcript levels. qRT-PCR revealed significantly lower levels of CDH1 expression in primary tumors with FOXA2 mutations. Our findings in vitro and in vivo suggest that reduced transcriptional activity associated with FOXA2 mutations in uterine cancers is likely to contribute to protumorigenic changes in gene expression.

Two Novel C-Terminal Frame shift Mutations in the CYB5R3 Gene Lead to Global Growth and Developmental Delay Associated with Recessive Congenital Methemoglobinemia Type II

Two Novel C-Terminal Frame shift Mutations in the CYB5R3 Gene Lead to Global Growth and Developmental Delay Associated with Recessive Congenital Methemoglobinemia Type II

Two novel C-terminal frameshift mutations in the \u03b2-globin gene lead to rapid mRNA decay

BackgroundThe thalassemia syndromes are classified according to the globin chain or chains whose production is affected. β-thalassemias are caused by point mutations or, more rarely, deletions or insertions of a few nucleotides in the β-globin gene or its immediate flanking sequences. These mutations interfere with the gene function either at the transcriptional, translational or posttranslational level.MethodsTwo cases of Polish patients with hereditary hemolytic anemia suspected of thalassemia were studied. DNA sequencing and mRNA quantification were performed. Stable human cell lines which express wild-type HBB and mutated versions were used to verify that detected mutation are responsible for mRNA degradation.ResultsWe identified two different frameshift mutations positioned in the third exon of HBB. Both patients harboring these mutations present the clinical phenotype of thalassemia intermedia and showed dominant pattern of inheritance. In both cases the mutations do not generate premature stop codon. Instead, slightly longer protein with unnatural C-terminus could be produced. Interestingly, although detected mutations are not expected to induce NMD, the mutant version of mRNA is not detectable. Restoring of the open reading frame brought back the RNA to that of the wild-type level.ConclusionOur results show that a lack of natural stop codon due to the frameshift in exon 3 of β-globin gene causes rapid degradation of its mRNA and indicate existence of novel surveillance pathway.

A novel frameshift mutation in CX46 associated with hereditary dominant cataracts in a Chinese family.

To investigate the genetic mutations that are associated the hereditary autosomal dominant cataract in a Chinese family. A Chinese family consisting of 20 cataract patients (including 9 male and 11 female) and 2 unaffected individuals from 5 generations were diagnosed to be a typical autosomal dominant cataract pedigree. Genomic DNA samples were extracted from the peripheral blood cells of the participants in this pedigree. Exon sequence was used for genetic mutation screening. In silico analysis was used to study the structure characteristics of connexin 46 (CX46) mutant. Immunoblotting was conduceted for testing the expression of CX46. To determine the involved genetic mutations, 11 well-known cataract-associated genes (cryaa, cryab, crybb1, crybb2, crygc, crygd, Gja3, Gja8, Hsf4, Mip and Pitx3) were chosen for genetic mutation test by using exon sequencing. A novel cytosine insertion at position 1195 of CX46 cDNA (c.1194_1195ins C) was found in the samples of 5 tested cataract patients but not in the unaffected 2 individuals nor in normal controls, which resulted in 30 amino acids more extension in CX46C-terminus (cx46fs400) compared with the wild-type CX46. In silico protein structure analysis indicated that the mutant showed distinctive hydrophobicity and protein secondary structure compared with the wild-type CX46. The immunoblot results revealed that CX46 protein, which expressed in the aging cataract lens tissues, was absence in the proband lens. In contrast, CX50, alpha A-crystallin and alphaB-crystallin expressed equally in both proband and aging cataract tissues. Those results revealed that the cx46fs400 mutation could impair CX46 protein expression. The insertion of cytosine at position 1195 of CX46 cDNA is a novel mutation site that is associated with the autosomal dominant cataracts in this Chinese family. The C-terminal frameshift mutation is involved in regulating CX46 protein expression.

EIF2S3Mutations Associated with Severe X-Linked Intellectual Disability Syndrome MEHMO

Impairment of translation initiation and its regulation within the integrated stress response (ISR) and related unfolded-protein response has been identified as a cause of several multisystemic syndromes. Here, we link MEHMO syndrome, whose genetic etiology was unknown, to this group of disorders. MEHMO is a rare X-linked syndrome characterized by profound intellectual disability, epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity. We have identified a C-terminal frameshift mutation (Ile465Serfs) in the EIF2S3 gene in three families with MEHMO syndrome and a novel maternally inherited missense EIF2S3 variant (c.324T>A; p.Ser108Arg) in another male patient with less severe clinical symptoms. The EIF2S3 gene encodes the γ subunit of eukaryotic translation initiation factor 2 (eIF2), crucial for initiation of protein synthesis and regulation of the ISR. Studies in patient fibroblasts confirm increased ISR activation due to the Ile465Serfs mutation and functional assays in yeast demonstrate that the Ile465Serfs mutation impairs eIF2γ function to a greater extent than tested missense mutations, consistent with the more severe clinical phenotype of the Ile465Serfs male mutation carriers. Thus, we propose that more severe EIF2S3 mutations cause the full MEHMO phenotype, while less deleterious mutations cause a milder form of the syndrome with only a subset of the symptoms.

Defining retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations.

This scientific commentary refers to ‘Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations’, by Stam et al. (doi:10.1093/brain/aww217) . Cerebral small vessel diseases refer to pathological processes that affect the structure or function of small vessels of the brain, including arteries, arterioles, capillaries, and occasionally venules (Pantoni, 2010). While the majority of small vessel diseases are sporadic age-related conditions driven by a complex interaction between genetic and cardiovascular risk factors or cerebrovascular amyloid deposition, inherited (familial) cerebral small vessel diseases have been increasingly reported in recent years (Joutel and Faraci, 2014). Of these inherited diseases, CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) and Fabry’s disease are among the most prevalent (Pantoni, 2010). The list of other, rarely observed single-gene small vessel diseases, often with systemic manifestations outside the brain, seems to be steadily increasing, posing diagnostic challenges in clinical practice. One such disorder is the fatal autosomal dominant condition caused by mutations in TREX1 (three prime repair exonuclease 1). TREX1 encodes a 3′-5′ DNA exonuclease involved in clearing cytosolic nucleic acids (Richards et al. , 2007). The disease was originally reported under different names, including hereditary vascular retinopathy, cerebroretinal vasculopathy, and hereditary endotheliopathy with retinopathy, nephropathy, and stroke (HERNS), thought to represent distinct autosomal dominant neurovascular syndromes. The mapping of all three syndromes to a common locus on chromosome 3p21.1-p21.3 and the identification of pathogenic heterozygous C-terminal frame-shift mutations in TREX1 (Richards et al. , 2007 …

Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations.

Cerebroretinal vasculopathy, hereditary vascular retinopathy, and hereditary endotheliopathy, retinopathy, nephropathy and stroke are neurovascular syndromes initially described as distinct entities. Recently they were shown to be one disease caused by C-terminal frame-shift mutations in TREX1 , which was termed ‘retinal vasculopathy with cerebral leukodystrophy’. Here we defined the genetic and clinicopathologic spectrum of this clinically and pathophysiologically poorly characterized and frequently misdiagnosed fatal neurovascular disorder. We identified five different TREX1 mutations in 78 members from 11 unrelated families and by using a standardized study protocol we retrospectively reviewed and aggregated the associated clinical, neuroimaging, and pathology data. Findings were similar across mutations and families. Sixty-four mutation carriers had vascular retinopathy. Neuroimaging revealed (i) punctate, hyperintense, white matter lesions with or without nodular enhancement in 97% of them; (ii) rim-enhancing mass lesions in 84%; and (iii) calcifications in the white matter in 52%. Ninety per cent had clinical manifestations of brain disease, including focal neurological deficits (68%), migraine (59%), cognitive impairment (56%), psychiatric disturbances (42%), and seizures (17%). One mutation carrier had enhancing brain lesions and neurological features but unknown retinopathy status. Additional systemic features included liver disease (78%), anaemia (74%), nephropathy (61%), hypertension (60%), mild Raynaud’s phenomenon (40%), and gastro-intestinal bleeding (27%). Mean (± standard deviation) age at diagnosis was 42.9 ± 8.3 years and at death 53.1 ± 9.6 years. Pathological examination revealed systemic vasculopathy with luminal narrowing and multi-laminated basement membranes. The 13 mutation carriers without retinopathy or brain lesions were on average 8 years younger (mean age: 35.1 ± 10.6 years). Of them, 54% had mild Raynaud’s phenomenon, 42% had migraine, and 23% had psychiatric disturbances. Retinal vasculopathy with cerebral leukodystrophy is an autosomal dominant systemic small-vessel disease due to specific TREX1 mutations and clinically primarily characterized by (i) visual impairment from vascular retinopathy; and (ii) neurological decline and premature death due to progressive enhancing cerebral white matter lesions. Impaired liver and kidney function, anaemia sometimes associated with gastrointestinal bleeding, hypertension, migraine, and Raynaud’s phenomenon appear to be part of the clinical spectrum as well. Penetrance seems high. Because of the pathogenetic basis and the emerging clinical picture with systemic manifestations and conspicuous absence of leukodystrophy, we renamed the disease ‘retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations’. We propose diagnostic criteria to facilitate clinical recognition and future studies. * Abbreviations : AGS : Aicardi-Goutieres syndrome MC : mutation carrier RVCL(-S) : retinal vasculopathy with cerebral leukoencephalopathy (and systemic manifestations)

Frequent somatic reversion of KRT1 mutations in ichthyosis with confetti.

Widespread reversion of genetic disease is rare; however, such events are particularly evident in some skin disorders in which normal clones develop on a background of affected skin. We previously demonstrated that mutations in keratin 10 (KRT10) cause ichthyosis with confetti (IWC), a severe dominant disorder that is characterized by progressive development of hundreds of normal skin spots via revertant mosaicism. Here, we report on a clinical and histological IWC subtype in which affected subjects have red, scaly skin at birth, experience worsening palmoplantar keratoderma in childhood, and develop hundreds of normal skin spots, beginning at around 20 years of age, that increase in size and number over time. We identified a causal de novo mutation in keratin 1 (KRT1). Similar to IWC-causing KRT10 mutations, this mutation in KRT1 resulted in a C-terminal frameshift, replacing 22 C-terminal amino acids with an alternate 30-residue peptide. Mutant KRT1 caused partial collapse of the cytoplasmic intermediate filament network and mislocalized to the nucleus. As with KRT10 mutations causing IWC, reversion of KRT1 mutations occurred via mitotic recombination. Because reversion is not observed with other disease-causing keratin mutations, the results of this study implicate KRT1 and KRT10 C-terminal frameshift mutations in the high frequency of revertant mosaicism in IWC.

TREX1 C-terminal frameshift mutations in the systemic variant of retinal vasculopathy with cerebral leukodystrophy

Retinal vasculopathy with cerebral leukodystrophy (RVCL) is an adult-onset disorder caused by C-terminal heterozygous frameshift (fs) mutations in the human 3'-5' DNA exonuclease TREX1. Hereditary systemic angiopathy (HSA) is considered a variant of RVCL with systemic involvement of unknown genetic cause, described in a unique family so far. Here we describe the second case of RVCL with systemic involvement, characterized by cerebral calcifications and pseudotumoral lesions, retinopathy, osteonecrosis, renal and hepatic failure. The genetic screening of TREX1 in this patient revealed the novel heterozygous T270fs mutation on the C-terminal region. On the same gene, we found the V235fs mutation, formerly shown in RVCL, in one patient previously reported with HSA. These mutations lead to important alterations of the C-terminal of the protein, with the loss of the transmembrane helix (T270fs) and the insertion of a premature stop codon, resulting in a truncated protein (V235fs). Functional analysis of T270fs-mutated fibroblasts showed a prevalent localization of the protein in the cytosol, rather than in the perinuclear region. RVCL with systemic involvement is an extremely rare condition, whose diagnosis is complex due to multiorgan manifestations, unusual radiological and histopathological findings, not easily attributable to a single disease. It should be suspected in young adults with systemic microangiopathy involving retina, liver, kidney, bones and brain. Here we confirm the causative role played by TREX1 autosomal dominant fs mutations disrupting the C-terminal of the protein, providing a model for the study of stroke in young adults.

Twenty patients including 7 probands with autosomal dominant cutis laxa confirm clinical and molecular homogeneity

BackgroundElastin gene mutations have been associated with a variety of phenotypes. Autosomal dominant cutis laxa (ADCL) is a rare disorder that presents with lax skin, typical facial characteristics, inguinal hernias, aortic root dilatation and pulmonary emphysema. In most patients, frameshift mutations are found in the 3’ region of the elastin gene (exons 30-34) which result in a C-terminally extended protein, though exceptions have been reported.MethodsWe clinically and molecularly characterized the thus far largest cohort of ADCL patients, consisting of 19 patients from six families and one sporadic patient.ResultsMolecular analysis showed C-terminal frameshift mutations in exon 30, 32, and 34 of the elastin gene and identified a mutational hotspot in exon 32 (c.2262delA). This cohort confirms the previously reported clinical constellation of skin laxity (100%), inguinal hernias (51%), aortic root dilatation (55%) and emphysema (37%).ConclusionADCL is a clinically and molecularly homogeneous disorder, but intra- and interfamilial variability in the severity of organ involvement needs to be taken into account. Regular cardiovascular and pulmonary evaluations are imperative in the clinical follow-up of these patients.

A Novel Degradation Signal Derived from Distal C-terminal Frameshift Mutations of KCNQ2 Protein Which Cause Neonatal Epilepsy

Benign familial neonatal convulsions is an autosomal-dominant idiopathic form of epilepsy primarily caused by gene mutations of the voltage-gated Kv7.2/KCNQ2/M-channel that exert only partial dominant-negative effects. However, the mechanism underlying the incomplete dominance of channel mutations, which cause epilepsy in infancy, remains unknown. Using mutagenesis and biochemistry combined with electrophysiology, we identified a novel degradation signal derived from distal C-terminal frameshift mutations, which impairs channel function. This degradation signal, transferable to non-channel CD4, can lead to accelerated degradation of mutant proteins through ubiquitin-independent proteasome machinery but does not affect mRNA quantity and protein trafficking. Functional dissection of this signal has revealed a key five-amino acid (RCXRG) motif critical for degradation. Taken together, our findings reveal a mechanism by which proteins that carry this signal are subject to degradation, leading to M-current dysfunction, which causes epilepsy.

Abstract B69: Development of a screening strategy for detecting rituximab-resistant mutations and characterization of mechanisms for acquiring resistant phenotype.

Abstract Background: Previously we reported that gene mutations of CD20 were found in patients with B-cell non-Hodgkin's lymphoma, and we proposed that C-terminal deletion mutations of CD20 might be related to relapse/resistance after rituximab therapy. Most of the cases that had these mutations were diagnosed as CD20 negative by immunohistochemical analysis using L26 monoclonal antibody. L26 recognizes the cytoplasmic region of CD20 molecules, but no more detailed information about its epitope had been reported. To make this clear, we determined the binding site of L26 antibody on CD20 protein. In addition, we developed novel antibodies that recognize wide variety of CD20 molecular subtypes including those having mutations. Methods: To determine the binding site of L26 antibody on CD20, we established six sub-lines that expressed a various length of C-terminal truncated CD20 using an originally CD20 negative myeloma cell line. These cell lines were used for epitope-mapping for L26 antibody. To detect comprehensive CD20 molecules including CD20 having mutation in C-terminal region, we developed antibodies that recognize near the amino terminal of CD20 molecules. Using these antibodies, we re-screened the specimens of CD20 negative cases determined by L26-based immunohistochemistry. Results: The epitope analysis revealed that L26 recognizes near the C-terminus of CD20 cytoplasmic region. This result showed that most of CD20 molecules with the C-terminal deletion mutation and frame-shift mutation could not be recognized by L26. So we screened previously diagnosed specimens and found several cases that included the cells stained by the N-terminal antibody but not by L26. Genetic analysis revealed that these cells had a mutation in the coding region of CD20. One of these cases, we successfully analyzed the phenotype of lymphoma cells having mutated CD20 in detail using cryopreserves living specimens. In this case, a frame shift mutation occurred due to one base nucleotide deletion, resulting in the translation of peptide of another reading frame of 41 amino acids after the amino acid position 250 with a slight early termination. Interestingly, although mutant CD20 molecule expressed adjacent to the plasma membrane, rituximab could not bind to the cells. DNA sequence analysis about genome and mRNA of CD20 gene indicated that the lymphoma cells of this patient had one mutated allele among two CD20 alleles. Discussions: The C-terminal region of CD20 undertakes a critical role in presentation of the large loop where the rituximab binding site locates. Thus, deletion or frame-shift mutation of C-terminal region impairs the antigenicity against rituximab and it may cause resistance to rituximab therapy. We propose here that finding a mutation by screening using antibodies of two kinds of epitope is useful in detection of irreversible resistant mutation for molecular targeted drug including rituximab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B69.

A Large Mutational Study in Pachyonychia Congenita

Pachyonychia congenita (PC) is a rare autosomal dominant skin disorder characterized predominantly by nail dystrophy and painful palmoplantar keratoderma. Additional clinical features include oral leukokeratosis, follicular keratosis, and cysts (steatocysts and pilosebaceous cysts). PC is due to heterozygous mutations in one of four keratin genes, namely, KRT6A, KRT6B, KRT16, or KRT17. Here, we report genetic analysis of 90 new families with PC in which we identified mutations in KRT6A, KRT6B, KRT16, or KRT17, thereby confirming their clinical diagnosis. A total of 21 previously unreported and 22 known mutations were found. Approximately half of the kindreds had mutations in KRT6A (52%), 28% had mutations in KRT16, 17% in KRT17, and 3% of families had mutations in KRT6B. Most of the mutations were heterozygous missense or small in-frame insertion/deletion mutations occurring within one of the helix boundary motif regions of the keratin polypeptide. More unusual mutations included heterozygous splice site mutations, nonsense mutations, and a 1-bp insertion mutation, leading to a frameshift and premature termination codon. This study, together with previously reported mutations, identifies mutation hotspot codons that may be useful in the development of personalized medicine for PC.