Missense Changes Research Articles

Factor VII deficiency: Unveiling the cellular and molecular mechanisms underlying three model alterations of the enzyme catalytic domain

Activated factor (F) VII is a vitamin K-dependent glycoprotein that initiates blood coagulation upon interaction with tissue factor. FVII deficiency is the most common of the rare congenital bleeding disorders. While the mutational pattern has been extensively characterized, the pathogenic molecular mechanisms of mutations, particularly at the intracellular level, have been poorly defined. Here, we aimed at elucidating the mechanisms underlying altered FVII biosynthesis in the presence of three mutation types in the catalytic domain: a missense change, a microdeletion and a frameshift/elongation, associated with severe or moderate to severe phenotypes. Using CHO-K1 cells transiently transfected with expression vectors containing the wild-type FVII cDNA (FVIIwt) or harboring the p.I289del, p.G420V or p.A354V-p.P464Hfs mutations, we found that the secretion of the FVII mutants was severely decreased compared to FVIIwt. The synthesis rate of the mutants was slower than the FVIIwt and delayed, and no degradation of the FVII mutants by proteasomes, lysosomes or cysteine proteases was observed. Confocal immunofluorescence microscopy studies showed that FVII variants were localized into the endoplasmic reticulum (ER) but were not detectable within the Golgi apparatus. These findings suggested that a common pathogenic mechanism, possibly a defective folding of the mutant proteins, was triggered by the FVII mutations. The misfolded state led to impaired trafficking of these proteins causing ER retention, which would explain the low to very low FVII plasma levels observed in patients carrying these mutations.

Factor VII deficiency: a novel missense variant and genotype\u2013phenotype correlation in patients from Southern Italy

This study aimed at attempting to correlate genotype and phenotype in factor VII deficiency. Here, we present molecular and clinical findings of 10 patients with factor VII deficiency. From 2013 to 2016, 10 subjects were referred to our center because of a prolonged prothrombin time identified during routine or presurgery examinations or after a laboratory assessment of a bleeding episode. Mutation characterization was performed using the bioinformatics applications PROMO, SIFT, and Polyphen-2. Structural changes in the factor VII protein were analyzed using the SPDB viewer tool. Of the 10 variants we identified, 1 was responsible for a novel missense change (c.1199G>C, p.Cys400Ser); in 2 cases we identified the c.-54G>A and c.509G>A (p.Arg170His) polymorphic variants in the 5′-upstream region of the factor VII gene and exon 6, respectively. To our knowledge, neither of these polymorphic variants has been described previously in factor VII-deficient patients. In silico predictions showed differences in binding sites for transcription factors caused by the c.-54G>A variant and a probable damaging effect of the p.Cys400Ser missense change on factor VII active conformation, leading to breaking of the Cys400-Cys428 disulfide bridge. Our findings further suggest that, independently of factor VII levels and of variants potentially affecting factor VII levels, environmental factors, e.g., trauma, could heavily influence the clinical phenotype of factor VII-deficient patients.

A novel de novo dominant mutation in ISCU associated with mitochondrial myopathy

BackgroundHereditary myopathy with lactic acidosis and myopathy with deficiency of succinate dehydrogenase and aconitase are variants of a recessive disorder characterised by childhood-onset early fatigue, dyspnoea and palpitations on trivial...

Functional Characterization of Rare RAB12 Variants and Their Role in Musician's and Other Dystonias.

Mutations in RAB (member of the Ras superfamily) genes are increasingly recognized as cause of a variety of disorders including neurological conditions. While musician’s dystonia (MD) and writer’s dystonia (WD) are task-specific movement disorders, other dystonias persistently affect postures as in cervical dystonia. Little is known about the underlying etiology. Next-generation sequencing revealed a rare missense variant (c.586A>G; p.Ile196Val) in RAB12 in two of three MD/WD families. Next, we tested 916 additional dystonia patients; 512 Parkinson’s disease patients; and 461 healthy controls for RAB12 variants and identified 10 additional carriers of rare missense changes among dystonia patients (1.1%) but only one carrier in non-dystonic individuals (0.1%; p = 0.005). The detected variants among index patients comprised p.Ile196Val (n = 6); p.Ala174Thr (n = 3); p.Gly13Asp; p.Ala148Thr; and p.Arg181Gln in patients with MD; cervical dystonia; or WD. Two relatives of MD patients with WD also carried p.Ile196Val. The two variants identified in MD patients (p.Ile196Val; p.Gly13Asp) were characterized on endogenous levels in patient-derived fibroblasts and in two RAB12-overexpressing cell models. The ability to hydrolyze guanosine triphosphate (GTP), so called GTPase activity, was increased in mutants compared to wildtype. Furthermore, subcellular distribution of RAB12 in mutants was altered in fibroblasts. Soluble Transferrin receptor 1 levels were reduced in the blood of all three tested p.Ile196Val carriers. In conclusion, we demonstrate an enrichment of missense changes among dystonia patients. Functional characterization revealed altered enzyme activity and lysosomal distribution in mutants suggesting a contribution of RAB12 variants to MD and other dystonias.

Novel nonsense gain-of-function NFKB2 mutations associated with a combined immunodeficiency phenotype

AbstractNF-κB signaling through its NFKB1-dependent canonical and NFKB2-dependent noncanonical pathways plays distinctive roles in a diverse range of immune processes. Recently, mutations in these 2 genes have been associated with common variable immunodeficiency (CVID). While studying patients with genetically uncharacterized primary immunodeficiencies, we detected 2 novel nonsense gain-of-function (GOF) NFKB2 mutations (E418X and R635X) in 3 patients from 2 families, and a novel missense change (S866R) in another patient. Their immunophenotype was assessed by flow cytometry and protein expression; activation of canonical and noncanonical pathways was examined in peripheral blood mononuclear cells and transfected HEK293T cells through immunoblotting, immunohistochemistry, luciferase activity, real-time polymerase chain reaction, and multiplex assays. The S866R change disrupted a C-terminal NF-κΒ2 critical site affecting protein phosphorylation and nuclear translocation, resulting in CVID with adrenocorticotropic hormone deficiency, growth hormone deficiency, and mild ectodermal dysplasia as previously described. In contrast, the nonsense mutations E418X and R635X observed in 3 patients led to constitutive nuclear localization and activation of both canonical and noncanonical NF-κΒ pathways, resulting in a combined immunodeficiency (CID) without endocrine or ectodermal manifestations. These changes were also found in 2 asymptomatic relatives. Thus, these novel NFKB2 GOF mutations produce a nonfully penetrant CID phenotype through a different pathophysiologic mechanism than previously described for mutations in NFKB2.

EC.O.1 - Genetic and histological characterisation of excitation-contraction coupling related structural myopathy cohort

Tubular aggregates and cylindrical spirals appear to be related entities, both arising from the sarcoplasmic reticulum, and sometimes appearing in the same biopsy. Tubular aggregates are inclusions of regular arrays of membrane tubules, while cylindrical spirals are accumulations of spiral lamellae around a central core. Although these have been reported as secondary findings in a range of conditions, some pure genetic forms of myopathy are characterised by the presence of tubular aggregates or cylindrical spirals. Tubular aggregate myopathy typically presents in childhood, causing slowly progressive weakness and cramps. Cylindrical spiral myopathy variably causes weakness, myalgia and exertional cramps, with onset in adulthood. Both diseases are dominant. The scarcity of cases impedes genetic and functional investigation. We have a cohort of two families and 10 isolated probands with tubular aggregate myopathy or cylindrical spirals. They underwent histological investigation, electron microscopy and next-generation DNA sequencing. Tubular aggregate myopathy genetic findings include known ORAI1 mutation c.G325A, p.V109M, and a novel putative STIM1 mutation c.A356G:p.D119G, and other candidate genes. Compound heterozygous TTN variants c.28654C>T, p.Q9552* and c.A15925G, p.I5309V were found in a cylindrical spiral myopathy case. The TTN missense change is predicted to affect splicing. Functional characterisation is ongoing, including mutant cell lines, cDNA analysis and fluorescence-based calcium handling assays. Here, we describe the work to date on characterising this cohort, including the histological and genetic features.

Characterisation of the novel deleterious RAD51C p.Arg312Trp variant and prioritisation criteria for functional analysis of RAD51C missense changes

Background:Despite a high prevalence of deleterious missense variants, most studies of RAD51C ovarian cancer susceptibility gene only provide in silico pathogenicity predictions of missense changes. We identified a novel deleterious RAD51C missense variant (p.Arg312Trp) in a high-risk family, and propose a criteria to prioritise RAD51C missense changes qualifying for functional analysis.Methods:To evaluate pathogenicity of p.Arg312Trp variant we used sequence homology, loss of heterozygosity (LOH) and segregation analysis, and a comprehensive functional characterisation. To define a functional-analysis prioritisation criteria, we used outputs for the known functionally confirmed deleterious and benign RAD51C missense changes from nine pathogenicity prediction algorithms.Results:The p.Arg312Trp variant failed to correct mitomycin and olaparib hypersensitivity and to complement abnormal RAD51C foci formation according to functional assays, which altogether with LOH and segregation data demonstrated deleteriousness. Prioritisation criteria were based on the number of predictors providing a deleterious output, with a minimum of 5 to qualify for testing and a PredictProtein score greater than 33 to assign high-priority indication.Conclusions:Our study points to a non-negligible number of RAD51C missense variants likely to impair protein function, provides a guideline to prioritise and encourage their selection for functional analysis and anticipates that reference laboratories should have available resources to conduct such assays.

Characterization of a variant of gap junction protein α8 identified in a family with hereditary cataract.

PurposeCongenital cataracts occur in isolation in about 70% of cases or are associated with other abnormalities such as anterior segment dysgenesis and microphthalmia. We identified a three-generation family in the University of California San Francisco glaucoma clinic comprising three individuals with congenital cataracts and aphakic glaucoma, one of whom also had microphthalmia. The purpose of this study was to identify a possible causative mutation in this family and to investigate its pathogenesis.MethodsWe performed exome sequencing and identified a putative mutation in gap junction protein α8 (GJA8). We used PCR and DNA sequencing of GJA8 in affected and unaffected members of the pedigree to test segregation of the variant with the phenotype. We tested cellular distribution and function of the variant protein by immunofluorescence and intercellular transfer of Neurobiotin in transiently transfected HeLa cells.ResultsExome sequencing revealed a variant in GJA8 (c.658A>G) encoding connexin50 (Cx50) that resulted in a missense change (p.N220D) in transmembrane domain 4. The variant was present in all three affected family members, but was also present in the proband's grandfather who was reported to be unaffected. The mutant protein localized to the plasma membrane and supported intercellular Neurobiotin transfer in HeLa cells.ConclusionsWe identified a variant in transmembrane domain 4 of Cx50 in a family with autosomal dominant congenital cataracts. This variant has been previously identified in other cataract cohorts, but it is also present in unaffected individuals. Our study demonstrates that the mutant protein localized to the plasma membrane and formed functional intercellular channels. These data suggest that GJA8 c.658A>G is most likely a benign rare variant.

Biallelic mutations in the homeodomain of NKX6-2 underlie a severe hypomyelinating leukodystrophy.

Hypomyelinating leukodystrophies are genetically heterogeneous disorders with overlapping clinical and neuroimaging features reflecting variable abnormalities in myelin formation. We report on the identification of biallelic inactivating mutations in NKX6-2, a gene encoding a transcription factor regulating multiple developmental processes with a main role in oligodendrocyte differentiation and regulation of myelin-specific gene expression, as the cause underlying a previously unrecognized severe variant of hypomyelinating leukodystrophy. Five affected subjects (three unrelated families) were documented to share biallelic inactivating mutations affecting the NKX6-2 homeobox domain. A trio-based whole exome sequencing analysis in the first family detected a homozygous frameshift change [c.606delinsTA; p.(Lys202Asnfs*?)]. In the second family, homozygosity mapping coupled to whole exome sequencing identified a homozygous nucleotide substitution (c.565G>T) introducing a premature stop codon (p.Glu189*). In the third family, whole exome sequencing established compound heterozygosity for a non-conservative missense change affecting a key residue participating in DNA binding (c.599G>A; p.Arg200Gln) and a nonsense substitution (c.589C>T; p.Gln197*), in both affected siblings. The clinical presentation was homogeneous, with four subjects having severe motor delays, nystagmus and absent head control, and one individual showing gross motor delay at the age of 6 months. All exhibited neuroimaging that was consistent with hypomyelination. These findings define a novel, severe form of leukodystrophy caused by impaired NKX6-2 function.

A mutation in Nischarin causes otitis media via LIMK1 and NF-κB pathways.

Otitis media (OM), inflammation of the middle ear (ME), is a common cause of conductive hearing impairment. Despite the importance of the disease, the aetiology of chronic and recurrent forms of middle ear inflammatory disease remains poorly understood. Studies of the human population suggest that there is a significant genetic component predisposing to the development of chronic OM, although the underlying genes are largely unknown. Using N-ethyl-N-nitrosourea mutagenesis we identified a recessive mouse mutant, edison, that spontaneously develops a conductive hearing loss due to chronic OM. The causal mutation was identified as a missense change, L972P, in the Nischarin (NISCH) gene. edison mice develop a serous or granulocytic effusion, increasingly macrophage and neutrophil rich with age, along with a thickened, inflamed mucoperiosteum. We also identified a second hypomorphic allele, V33A, with only modest increases in auditory thresholds and reduced incidence of OM. NISCH interacts with several proteins, including ITGA5 that is thought to have a role in modulating VEGF-induced angiogenesis and vascularization. We identified a significant genetic interaction between Nisch and Itga5; mice heterozygous for Itga5-null and homozygous for edison mutations display a significantly increased penetrance and severity of chronic OM. In order to understand the pathological mechanisms underlying the OM phenotype, we studied interacting partners to NISCH along with downstream signalling molecules in the middle ear epithelia of edison mouse. Our analysis implicates PAK1 and RAC1, and downstream signalling in LIMK1 and NF-κB pathways in the development of chronic OM.

Novel SOX17 frameshift mutations in endometrial cancer are functionally distinct from recurrent missense mutations.

Extensive genomic profiling for endometrioid endometrial carcinoma (EEC) has pointed to genes and pathways important in uterine development as critical mediators of endometrial tumorigenesis. SOX17 is a developmental transcription factor necessary for proper endoderm formation that has been implicated as a tumor suppressor and shown to modulate WNT signaling. SOX17 mutation analysis in 539 primary EECs revealed frequent missense and frameshift mutations with an overall 11.5% mutation rate. More than half the mutations identified were frameshifts (32 of 62), and the hotspot missense changes, p.Ala96Gly and p.Ser403Ile, were seen in 14 tumors. None of the cases with a mutation had a second SOX17 mutation or evidence of allelic loss. Immunofluorescence microscopy performed on primary samples showed that there were no changes in SOX17 protein expression associated with mutation. Low/absent SOX17 staining was significantly associated with advanced stage, high tumor grade and reduced recurrence-free survival. Functional assessment of the two hotspot missense mutations and three representative frameshift mutations showed that SOX17-A96G and SOX17-S403I have transcriptional activities similar to SOX17 wild-type (WT), whereas none of the frameshift mutant proteins showed transcriptional activity. Forced expression of SOX17-WT, -A96G or -S403I in EC cell lines moderately increased β-catenin mediated transcription, which contrasts with previous data showing SOX17 is an inhibitor of TCF/β-catenin signaling. The proliferation of EC cell lines was expectedly reduced by transfection with SOX17-WT, and further reduced by SOX17-A96G and SOX17-S403I. These data implicate SOX17 mutation as a selected event in EEC, with clear differences between the missense and frameshift mutations.

Abstract 1282: Analysis of missense variants in BRCA1 BRCT domains

Abstract Currently, only ~¼ (133/600) of documented single nucleotide variation leading to missense changes in the BRCA1 gene have been characterized as pathogenic or non-pathogenic (IARC Classes 4 & 5 and 1 & 2, respectively) posing a challenge for carriers of these variants. A detailed understanding of structure-function relationships is critical for the assessment of these variants in the interpretation of data from functional assay and for the development of functional impact predictors. Thus, we have systematically probed the C-terminus of BRCA1 including the N-terminal border of the BRCT domains (M1650, S1651), the BRCT linker region (G1738, D1739, V1740, G1743, H1746, G1748, P1749, R1753, and D1757), and the alpha 1 and alpha’1 helices in BRCT[N] and [C], respectively. We assessed the functional impact of 47 missense variants using a validated transcriptional assay by a protein fusion of the GAL4 DNA-binding domain to BRCA1 C-terminus (aa 1,396-1,863) in HEK 293FT cells. Analysis of functional data redefined the BRCT border (previously thought to be at S1651) at residue M1650. Overall, mutations in the BRCT linker had severe impact on the function. Mutations in the alpha1 and alpha’1 helices also tended to display impaired function despite the lack of functional impact of 16 out of the 17 variants previously analyzed. In summary, our study provides increased granularity in our understanding of BRCA1 regions less tolerant to changes and therefore more likely to contain pathogenic variants. This knowledge will be critical for the assignment of different prior probabilities of pathogenicity given the variant location, improving risk assessment and clinical management of carriers of BRCA1 missense alleles Note: This abstract was not presented at the meeting. Citation Format: Giuliano Di Pietro, Vanessa Camara Fernandes, Kwabena Amankwah, Cara Shields, Volha A. Golubeva, Carly Harro, Marcelo Alex Carvalho, Alvaro N. Monteiro. Analysis of missense variants in BRCA1 BRCT domains [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1282. doi:10.1158/1538-7445.AM2017-1282

Mutation Spectrum in the CACNA1A Gene in 49 Patients with Episodic Ataxia

Episodic ataxia is an autosomal dominant ion channel disorder characterized by episodes of imbalance and incoordination. The disease is genetically heterogeneous and is classified as episodic ataxia type 2 (EA2) when it is caused by a mutation in the CACNA1A gene, encoding the α1A subunit of the P/Q-type voltage-gated calcium channel Cav2.1. The vast majority of EA2 disease-causing variants are loss-of-function (LoF) point changes leading to decreased channel currents. CACNA1A exonic deletions have also been reported in EA2 using quantitative approaches. We performed a mutational screening of the CACNA1A gene, including the promoter and 3′UTR regions, in 49 unrelated patients diagnosed with episodic ataxia. When pathogenic variants were not found by sequencing, we performed a copy number variant (CNV) analysis to screen for duplications or deletions. Overall, sequencing screening allowed identification of six different point variants (three nonsense and three missense changes) and two coding indels, one of them found in two unrelated patients. Additionally, CNV analysis identified a deletion in a patient spanning exon 35 as a result of a recombination event between flanking intronic Alu sequences. This study allowed identification of potentially pathogenic alterations in our sample, five of them novel, which cover 20% of the patients (10/49). Our data suggest that most of these variants are disease-causing, although functional studies are required.

MS2 Lysis of Escherichia coli Depends on Host Chaperone DnaJ.

The L protein of the single-stranded RNA phage MS2 causes lysis of Escherichia coli without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (insensitivity to Llysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the dnaJP330Q mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the dnaJP330Q mutant was recessive and could be suppressed by Lovercomes dnaJ (Lodj ) alleles selected for restoration of lysis. All three Lodj alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ∼20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the Lodj truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of ϕX174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions.IMPORTANCE Small single-stranded nucleic acid lytic phages (Microviridae and Leviviridae) lyse their host by expressing a single "protein antibiotic." The protein antibiotics from two out of three prototypic small lytic viruses have been shown to inhibit two different steps in the conserved PG biosynthesis pathway. However, the molecular basis of lysis caused by L, the lysis protein of the third prototypic virus, MS2, is unknown. The significance of our research lies in the identification of DnaJ as a chaperone in the MS2 L lysis pathway and the identification of the minimal lytic domain of MS2 L. Additionally, our research highlights the importance of the highly conserved P330 residue in the C-terminal domain of DnaJ for specific protein interactions.

Not only dominant, not only optic atrophy: expanding the clinical spectrum associated with OPA1 mutations

BackgroundHeterozygous mutations in OPA1 are a common cause of autosomal dominant optic atrophy, sometimes associated with extra-ocular manifestations. Few cases harboring compound heterozygous OPA1 mutations have been described manifesting complex neurodegenerative disorders in addition to optic atrophy.ResultsWe report here three patients: one boy showing an early-onset mitochondrial disorder with hypotonia, ataxia and neuropathy that was severely progressive, leading to early death because of multiorgan failure; two unrelated sporadic girls manifesting a spastic ataxic syndrome associated with peripheral neuropathy and, only in one, optic atrophy. Using a targeted resequencing of 132 genes associated with mitochondrial disorders, in two probands we found compound heterozygous mutations in OPA1: in the first a 5 nucleotide deletion, causing a frameshift and insertion of a premature stop codon (p.Ser64Asnfs*7), and a missense change (p.Ile437Met), which has recently been reported to have clinical impact; in the second, a novel missense change (p.Val988Phe) co-occurred with the p.Ile437Met substitution. In the third patient a homozygous mutation, c.1180G > A (p.Ala394Thr) in OPA1 was detected by a trio-based whole exome sequencing approach. One of the patients presented also variants in mitochondrial DNA that may have contributed to the peculiar phenotype.The deleterious effect of the identified missense changes was experimentally validated in yeast model. OPA1 level was reduced in available patients’ biological samples, and a clearly fragmented mitochondrial network was observed in patients’ fibroblasts.ConclusionsThis report provides evidence that bi-allelic OPA1 mutations may lead to complex and severe multi-system recessive mitochondrial disorders, where optic atrophy might not represent the main feature.

Genotype and phenotype spectrum of NRAS germline variants.

RASopathies comprise a group of disorders clinically characterized by short stature, heart defects, facial dysmorphism, and varying degrees of intellectual disability and cancer predisposition. They are caused by germline variants in genes encoding key components or modulators of the highly conserved RAS-MAPK signalling pathway that lead to dysregulation of cell signal transmission. Germline changes in the genes encoding members of the RAS subfamily of GTPases are rare and associated with variable phenotypes of the RASopathy spectrum, ranging from Costello syndrome (HRAS variants) to Noonan and Cardiofaciocutaneous syndromes (KRAS variants). A small number of RASopathy cases with disease-causing germline NRAS alterations have been reported. Affected individuals exhibited features fitting Noonan syndrome, and the observed germline variants differed from the typical oncogenic NRAS changes occurring as somatic events in tumours. Here we describe 19 new cases with RASopathy due to disease-causing variants in NRAS. Importantly, four of them harbored missense changes affecting Gly12, which was previously described to occur exclusively in cancer. The phenotype in our cohort was variable but well within the RASopathy spectrum. Further, one of the patients (c.35G>A; p.(Gly12Asp)) had a myeloproliferative disorder, and one subject (c.34G>C; p.(Gly12Arg)) exhibited an uncharacterized brain tumour. With this report, we expand the genotype and phenotype spectrum of RASopathy-associated germline NRAS variants and provide evidence that NRAS variants do not spare the cancer-associated mutation hotspots.

TBC1D24 Mutations in a Sibship with Multifocal Polymyoclonus.

BackgroundAdvances in molecular genetic technologies have improved our understanding of genetic causes of rare neurological disorders with features of myoclonus.Case ReportA family with two affected siblings, presenting with multifocal polymyoclonus and neurodevelopmental delay, was recruited for whole-exome sequencing following unyielding diagnostic neurometabolic investigations. Compound heterozygous mutations in TBC1D24, a gene previously associated with various epilepsy phenotypes and hearing loss, were identified in both siblings. The mutations included a missense change c.457G>A (p.Glu157Lys), and a novel frameshift mutation c.545del (p.Thr182Serfs*6).DiscussionWe propose that TBC1D24-related diseases should be in the differential diagnosis for children with polymyoclonus.

De novo REEP2 missense mutation in pure hereditary spastic paraplegia.

Alterations in proteins that regulate endoplasmic reticulum morphology are common causes of hereditary spastic paraplegia (SPG1‐78, plus others). Mutations in the REEP1 gene that encodes an endoplasmic reticulum‐shaping protein are well‐known causes of SPG31, a common autosomal dominant spastic paraplegia. A closely‐related gene, REEP2, is mutated in SPG72, with both autosomal and recessive inheritances. Here, we report a patient with a pure hereditary spastic paraplegia due to a de novo missense mutation (c.119T > G, p.Met40Arg) in REEP2 at a highly‐conserved residue very close to another known pathogenic missense change. This represents only the second autosomal dominant SPG72 missense mutation reported.

Exome sequencing reveals a de novo POLD1 mutation causing phenotypic variability in mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL)

BackgroundMandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL) is an autosomal dominant systemic disorder characterized by prominent loss of subcutaneous fat, a characteristic facial appearance and metabolic abnormalities. This syndrome is caused by heterozygous de novo mutations in the POLD1 gene. To date, 19 patients with MDPL have been reported in the literature and among them 14 patients have been characterized at the molecular level. Twelve unrelated patients carried a recurrent in-frame deletion of a single codon (p.Ser605del) and two other patients carried a novel heterozygous mutation in exon 13 (p.Arg507Cys). Additionally and interestingly, germline mutations of the same gene have been involved in familial polyposis and colorectal cancer (CRC) predisposition. Patients and MethodsWe describe a male and a female patient with MDPL respectively affected with mild and severe phenotypes. Both of them showed mandibular hypoplasia, a beaked nose with bird-like facies, prominent eyes, a small mouth, growth retardation, muscle and skin atrophy, but the female patient showed such a severe and early phenotype that a first working diagnosis of Hutchinson-Gilford Progeria was made. The exploration was performed by direct sequencing of POLD1 gene exon 15 in the male patient with a classical MDPL phenotype and by whole exome sequencing in the female patient and her unaffected parents. ResultsExome sequencing identified in the latter patient a de novo heterozygous undescribed mutation in the POLD1 gene (NM_002691.3: c.3209T>A), predicted to cause the missense change p.Ile1070Asn in the ZnF2 (Zinc Finger 2) domain of the protein. This mutation was not reported in the 1000 Genome Project, dbSNP and Exome sequencing databases. Furthermore, the Isoleucine1070 residue of POLD1 is highly conserved among various species, suggesting that this substitution may cause a major impairment of POLD1 activity. For the second patient, affected with a typical MDPL phenotype, direct sequencing of POLD1 exon 15 revealed the recurrent in-frame deletion (c.1812_1814del, p.S605del). ConclusionOur work highlights that mutations in different POLD1 domains can lead to phenotypic variability, ranging from dominantly inherited cancer predisposition syndromes, to mild MDPL phenotypes without lifespan reduction, to very severe MDPL syndromes with major premature aging features. These results also suggest that POLD1 gene testing should be considered in patients presenting with severe progeroid features.

Ataxic form of autosomal recessive PEX10-related peroxisome biogenesis disorders with a novel compound heterozygous gene mutation and characteristic clinical phenotype

Peroxisome biogenesis factor 10 (PEX10) is involved in the import of peroxisomal matrix proteins, and the mutation of this gene causes 3 subtypes of peroxisome biogenesis disorders, namely Zellweger syndrome (severe), neonatal adrenoleukodystrophy (moderate) and an ataxic form (mild). Here, we report 3 siblings of the ataxic form with cerebellar ataxia, mild mental retardation, and 3 additional characteristic features: mydriasis, hyperreflexia and involuntary head movement. All 3 siblings are compound heterozygous for a previously reported mutation, c.2T>C (p.M1T), and a novel mutation, c.920G>A, causing a missense change (p.C307Y) located in the RING finger domain of PEX10. The present cases suggest that these PEX10 mutations involve not only cerebellar but also more multiple nervous systems including pupillary autonomic, pyramidal and extrapyramidal systems.