“Mandibulofacial dysostosis with microcephaly” caused by EFTUD2 mutations: Expanding the phenotype

Heterozygous mutations in the EFTUD2 were identified in 12 individuals with a rare sporadic craniofacial condition termed Mandibulofacial dysostosis with microcephaly (MIM 610536). We present clinical and radiographic features of three additional patients with de novo heterozygous mutations in EFTUD2. Although clinical features overlap with findings of the original report (choanal atresia, cleft palate, maxillary and mandibular hypoplasia, and microtia), microcephaly was present in two of three patients and cognitive impairment was milder in those with head circumference proportional to height. Our cases expand the phenotypic spectrum to include epibulbar dermoids and zygomatic arch clefting. We suggest that craniofacial computed tomography studies to assess cleft of zygomatic arch may assist in making this diagnosis. We recommend consideration of EFTUD2 testing in individuals with features of oculo‐auriculo‐vertebral spectrum and bilateral microtia, or individuals with atypical CHARGE syndrome who do not have a CHD7 mutation, particularly those with a zygomatic arch cleft. The absence of microcephaly in one patient indicates that it is a highly variable phenotypic feature. © 2012 Wiley Periodicals, Inc.     

XPC branch‐point sequence mutations disrupt U2 snRNP binding,resulting in abnormal pre‐mRNA splicing in xeroderma pigmentosum patients

Mutations in two branch‐point sequences (BPS) in intron 3 of the XPC DNA repair gene affect pre‐mRNA splicing in association with xeroderma pigmentosum (XP) with many skin cancers (XP101TMA) or no skin cancer (XP72TMA), respectively. To investigate the mechanism of these abnormalities we now report that transfection of minigenes with these mutations revealed abnormal XPC pre‐mRNA splicing that mimicked pre‐mRNA splicing in the patients' cells. DNA oligonucleotide‐directed RNase H digestion demonstrated that mutations in these BPS disrupt U2 snRNP–BPS interaction. XP101TMA cells had no detectable XPC protein but XP72TMA had 29% of normal levels. A small amount of XPC protein was detected at sites of localized ultraviolet (UV)‐damaged DNA in XP72TMA cells which then recruited other nucleotide excision repair (NER) proteins. In contrast, XP101TMA cells had no detectable recruitment of XPC or other NER proteins. Post‐UV survival and photoproduct assays revealed greater reduction in DNA repair in XP101TMA cells than in XP72TMA. Thus mutations in XPC BPS resulted in disruption of U2 snRNP‐BPS interaction leading to abnormal pre‐mRNA splicing and reduced XPC protein. At the cellular level these changes were associated with features of reduced DNA repair including diminished NER protein recruitment, reduced post‐UV survival and impaired photoproduct removal. Hum Mutat 30:1–9, 2009. Published 2009 Wiley‐Liss, Inc.     

D‐2‐hydroxyglutaric aciduria Type I: Functional analysis of D2HGDH missense variants

D‐2‐hydroxyglutaric aciduria Type I (D‐2‐HGA Type I), a neurometabolic disorder with a broad clinical spectrum, is caused by recessive variants in the D2HGDH gene encoding D‐2‐hydroxyglutarate dehydrogenase (D‐2‐HGDH). We and others detected 42 potentially pathogenic variants in D2HGDH of which 31 were missense. We developed functional studies to investigate the effect of missense variants on D‐2‐HGDH catalytic activity. Site‐directed mutagenesis was used to introduce 31 missense variants in the pCMV5‐D2HGDH expression vector. The wild type and missense variants were overexpressed in HEK293 cells. D‐2‐HGDH enzyme activity was evaluated based on the conversion of [²H₄]D‐2‐HG to [²H₄]2‐ketoglutarate, which was subsequently converted into [²H₄]L‐glutamate and the latter quantified by LC‐MS/MS. Eighteen variants resulted in almost complete ablation of D‐2‐HGDH activity and thus, should be considered pathogenic. The remaining 13 variants manifested residual activities ranging between 17% and 94% of control enzymatic activity. Our functional assay evaluating the effect of novel D2HGDH variants will be beneficial for the classification of missense variants and determination of pathogenicity.     

Assessment of the functional impact on the pre‐mRNA splicing process of 28 nucleotide variants associated with Pompe disease in GAA exon 2 and their recovery using antisense technology

Glycogen storage disease II (GSDII), also called Pompe disease, is an autosomal recessive inherited disease caused by a defect in glycogen metabolism due to the deficiency of the enzyme acid alpha‐glucosidase (GAA) responsible for its degradation. So far, more than 500 sequence variants of the GAA gene have been reported but their possible involvement on the pre‐messenger RNA splicing mechanism has not been extensively studied. In this work, we have investigated, by an in vitro functional assay, all putative splicing variants within GAA exon 2 and flanking introns. Our results show that many variants falling in the canonical splice site or the exon can induce GAA exon 2 skipping. In these cases, therefore, therapeutic strategies aimed at restoring protein folding of partially active mutated GAA proteins might not be sufficient. Regarding this issue, we have tested the effect of antisense oligonucleotides (AMOs) that were previously shown capable of rescuing splicing misregulation caused by the common c.‐32‐13T>G variant associated with the childhood/adult phenotype of GSDII. Interestingly, our results show that these AMOs are also quite effective in rescuing the splicing impairment of several exonic splicing variants, thus widening the potential use of these effectors for GSDII treatment.     

Hermansky–Pudlak syndrome type 2: Aberrant pre‐mRNA splicing and mislocalization of granule proteins in neutrophils

Hermansky–Pudlak syndrome type 2 (HPS2) is a syndrome caused by mutations in the beta‐3A subunit of the adaptor protein (AP)‐3 complex (AP3B1 gene). We describe five unreported cases with four novel mutations, one of which caused aberrant pre‐mRNA splicing. A point mutation c.2702C>G in exon 23 of the AP3B1 gene caused deletion of 112 bp in the mRNA in two siblings. This mutation activates a cryptic donor splice site that overrules the wild‐type donor splice site of this exon. Three other novel mutations in AP3B1 were identified, that is, a nonsense mutation c.716G>A (p.Trp239Ter), a 1‐bp and a 4‐bp deletion c.177delA and c.1839_1842delTAGA, respectively, both causing frameshift and premature termination of translation. Mass spectrometry in four of these HPS2 patients demonstrated the (near) absence of all AP‐3 complex subunits. Immunoelectron microscopy on the neutrophils of two of these patients showed abnormal granule formation. We found clear mislocalization of myeloperoxidase in the neutrophils even though the content of this protein but not the activity seemed to be present at normal levels. In sum, HPS2 is the result of the absence of the entire AP‐3 complex, which results in severe neutropenia with a defect in granule formation as the major hematological finding.     

Genetic and bioinformatics analysis of four novel GCK missense variants detected in Caucasian families with GCK‐MODY phenotype

Heterozygous loss‐of‐function mutations in the glucokinase (GCK) gene cause maturity‐onset diabetes of the young (MODY) subtype GCK (GCK‐MODY/MODY2). GCK sequencing revealed 16 distinct mutations (13 missense, 1 nonsense, 1 splice site, and 1 frameshift‐deletion) co‐segregating with hyperglycaemia in 23 GCK‐MODY families. Four missense substitutions (c.718A>G/p.Asn240Asp, c.757G>T/p.Val253Phe, c.872A>C/p.Lys291Thr, and c.1151C>T/p.Ala384Val) were novel and a founder effect for the nonsense mutation (c.76C>T/p.Gln26*) was supposed. We tested whether an accurate bioinformatics approach could strengthen family‐genetic evidence for missense variant pathogenicity in routine diagnostics, where wet‐lab functional assays are generally unviable. In silico analyses of the novel missense variants, including orthologous sequence conservation, amino acid substitution (AAS)‐pathogenicity predictors, structural modeling and splicing predictors, suggested that the AASs and/or the underlying nucleotide changes are likely to be pathogenic. This study shows how a careful bioinformatics analysis could provide effective suggestions to help molecular‐genetic diagnosis in absence of wet‐lab validations.     

Functional and cellular localization diversity associated with Fukutin‐related protein patient genetic variants

Genetic variants in Fukutin‐related protein (FKRP), an essential enzyme of the glycosylation pathway of α‐dystroglycan, can lead to pathologies with different severities affecting the eye, brain, and muscle tissues. Here, we generate an in vitro cellular system to characterize the cellular localization as well as the functional potential of the most common FKRP patient missense mutations. We observe a differential retention in the endoplasmic reticulum (ER), the indication of misfolded proteins. We find data supporting that mutant protein able to overcome this ER‐retention through overexpression present functional levels comparable to the wild‐type. We also identify a specific region in FKRP protein localized between residues 300 and 321 in which genetic variants found in patients lead to correctly localized proteins but which are nevertheless functionally impaired or catalytically dead in our model, indicating that this particular region might be important for the enzymatic activity of FKRP within the Golgi. Our system thus allows the functional testing of patient‐specific mutant proteins and the identification of candidate mutants to be further explored with the aim of finding pharmacological treatments targeting the protein quality control system.     

Protein destabilization and loss of protein‐protein interaction are fundamental mechanisms in cblA‐type methylmalonic aciduria

Mutations in the human MMAA gene cause the metabolic disorder cblA‐type methylmalonic aciduria (MMA), although knowledge of the mechanism of dysfunction remains lacking. MMAA regulates the incorporation of the cofactor adenosylcobalamin (AdoCbl), generated from the MMAB adenosyltransferase, into the destination enzyme methylmalonyl‐CoA mutase (MUT). This function of MMAA depends on its GTPase activity, which is stimulated by an interaction with MUT. Here, we present 67 new patients with cblA‐type MMA, identifying 19 novel mutations. We biochemically investigated how missense mutations in MMAA in 22 patients lead to disease. About a third confer instability to the recombinant protein in bacterial and human expression systems. All 15 purified mutant proteins demonstrated wild‐type like intrinsic GTPase activity and only one (p.Asp292Val), where the mutation is in the GTP binding domain, revealed decreased GTP binding. However, all mutations strongly decreased functional association with MUT by reducing GTPase activity stimulation upon incubation with MUT, while nine mutant proteins additionally lost the ability to physically bind MUT. Finally, all mutations interfered with gating the transfer of AdoCbl from MMAB to MUT. This work suggests loss of functional interaction between MMAA and MUT as a disease‐causing mechanism that impacts processing and assembly of a cofactor to its destination enzyme.     

TEG-1 CD2BP2 regulates stem cell proliferation and sex determination in the C. elegans germ line and physically interacts with the UAF-1 U2AF65 splicing factor

Background : For a stem cell population to exist over an extended period, a balance must be maintained between self‐renewing (proliferating) and differentiating daughter cells. Within the Caenorhabditis elegans germ line, this balance is controlled by a genetic regulatory pathway, which includes the canonical Notch signaling pathway. Results : Genetic screens identified the gene teg‐1 as being involved in regulating the proliferation versus differentiation decision in the C. elegans germ line. Cloning of TEG‐1 revealed that it is a homolog of mammalian CD2BP2, which has been implicated in a number of cellular processes, including in U4/U6.U5 tri‐snRNP formation in the pre‐mRNA splicing reaction. The position of teg‐1 in the genetic pathway regulating the proliferation versus differentiation decision, its single mutant phenotype, and its enrichment in nuclei, all suggest TEG‐1 also functions as a splicing factor. TEG‐1, as well as its human homolog, CD2BP2, directly bind to UAF‐1 U2AF65, a component of the U2 auxiliary factor. Conclusions : TEG‐1 functions as a splicing factor and acts to regulate the proliferation versus meiosis decision. The interaction of TEG‐1 CD2BP2 with UAF‐1 U2AF65, combined with its previously described function in U4/U6.U5 tri‐snRNP, suggests that TEG‐1 CD2BP2 functions in two distinct locations in the splicing cascade. Developmental Dynamics 241:505–521, 2012.© 2012 Wiley Periodicals, Inc.     

Risks of breast and ovarian cancer for women harboring pathogenic missense variants in BRCA1 and BRCA2 compared with those harboring protein truncating variants

PurposeGermline genetic testing for BRCA1 and BRCA2 variants has been a part of clinical practice for >2 decades. However, no studies have compared the cancer risks associated with missense pathogenic variants (PVs) with those associated with protein truncating (PTC) variants.MethodsWe collected 582 informative pedigrees segregating 1 of 28 missense PVs in BRCA1 and 153 pedigrees segregating 1 of 12 missense PVs in BRCA2. We analyzed 324 pedigrees with PTC variants in BRCA1 and 214 pedigrees with PTC variants in BRCA2. Cancer risks were estimated using modified segregation analysis.ResultsEstimated breast cancer risks were markedly lower for women aged >50 years carrying BRCA1 missense PVs than for the women carrying BRCA1 PTC variants (hazard ratio [HR] = 3.9 [2.4-6.2] for PVs vs 12.8 [5.7-28.7] for PTC variants; P = .01), particularly for missense PVs in the BRCA1 C-terminal domain (HR = 2.8 [1.4-5.6]; P = .005). In case of BRCA2, for women aged >50 years, the HR was 3.9 (2.0-7.2) for those heterozygous for missense PVs compared with 7.0 (3.3-14.7) for those harboring PTC variants. BRCA1 p.[Cys64Arg] and BRCA2 p.[Trp2626Cys] were associated with particularly low risks of breast cancer compared with other PVs.ConclusionThese results have important implications for the counseling of at-risk women who harbor missense PVs in the BRCA1/2 genes.     

Functional interrogation of Lynch syndrome‐associated MSH2 missense variants via CRISPR‐Cas9 gene editing in human embryonic stem cells

Lynch syndrome (LS) predisposes patients to cancer and is caused by germline mutations in the DNA mismatch repair (MMR) genes. Identifying the deleterious mutation, such as a frameshift or nonsense mutation, is important for confirming an LS diagnosis. However, discovery of a missense variant is often inconclusive. The effects of these variants of uncertain significance (VUS) on disease pathogenesis are unclear, though understanding their impact on protein function can help determine their significance. Laboratory functional studies performed to date have been limited by their artificial nature. We report here an in‐cellulo functional assay in which we engineered site‐specific MSH2 VUS using clustered regularly interspaced short palindromic repeats‐Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild‐type gene. We characterized the impact of the variants on cellular MMR functions including DNA damage response signaling and the repair of DNA microsatellites. We classified the MMR functional capability of eight of 10 VUS providing valuable information for determining their likelihood of being bona fide pathogenic LS variants. This human cell‐based assay system for functional testing of MMR gene VUS will facilitate the identification of high‐risk LS patients.     

Damaging de novo missense variants in EEF1A2 lead to a developmental and degenerative epileptic‐dyskinetic encephalopathy

Heterozygous de novo variants in the eukaryotic elongation factor EEF1A2 have previously been described in association with intellectual disability and epilepsy but never functionally validated. Here we report 14 new individuals with heterozygous EEF1A2 variants. We functionally validate multiple variants as protein‐damaging using heterologous expression and complementation analysis. Our findings allow us to confirm multiple variants as pathogenic and broaden the phenotypic spectrum to include dystonia/choreoathetosis, and in some cases a degenerative course with cerebral and cerebellar atrophy. Pathogenic variants appear to act via a haploinsufficiency mechanism, disrupting both the protein synthesis and integrated stress response functions of EEF1A2. Our studies provide evidence that EEF1A2 is highly intolerant to variation and that de novo pathogenic variants lead to an epileptic‐dyskinetic encephalopathy with both neurodevelopmental and neurodegenerative features. Developmental features may be driven by impaired synaptic protein synthesis during early brain development while progressive symptoms may be linked to an impaired ability to handle cytotoxic stressors.     

Leukoencephalopathy in Al‐Raqad syndrome: Expanding the clinical and neuroimaging features caused by a biallelic novel missense variant in DCPS

Al‐Raqad syndrome (ARS) is a rare autosomal recessive congenital disorder, associated mainly with developmental delay, and intellectual disability. This syndrome is caused by mutations in DCPS, encoding scavenger mRNA decapping enzyme, which plays a role in the 3‐prime‐end mRNA decay pathway. Whole‐exome sequencing was performed on an offspring of a consanguineous family presenting with developmental delay, intellectual disability, growth retardation, mild craniofacial abnormalities, cerebral and cerebellar atrophy, and white matter diffuse hypomyelination pattern. A novel biallelic missense variant, c.918G>C p. (Glu306Asp), in the DCPS gene was identified which was confirmed by sanger sequencing and segregation analysis subsequently. Few cases of ARS have been described up to now, and this study represents a 7‐years‐old boy presenting with central and peripheral nervous system impaired myelination in addition to ocular and dental manifestation, therefore outstretch both neuroimaging and clinical findings of this ultra‐rare syndrome.     

Intronic variants in BRCA1 and BRCA2 that affect RNA splicing can be reliably selected by splice-site prediction programs

A large number of sequence variants identified in BRCA1 and BRCA2 cannot be distinguished as either disease-causing mutations or neutral variants. These so-called unclassified variants (UVs) include variants that are located in the intronic sequences of BRCA1 and BRCA2. The purpose of this study was to assess the use of splice-site prediction programs (SSPPs) to select intronic variants in BRCA1 and BRCA2 that are likely to affect RNA splicing. We performed in vitro molecular characterization of RNA of six intronic variants in BRCA1 and BRCA2. In four cases (BRCA1, c.81-6T>A and c.4986+5G>T; BRCA2, c.7617+2T>G and c.8754+5G>A) a deleterious effect on RNA splicing was seen, whereas the c.135-15_-12del variant in BRCA1 showed no effect on RNA splicing. In the case of the BRCA2 c.68-7T>A variant, RNA analysis was not sufficient to establish the clinical significance. Six SSPPs were used to predict whether an effect on RNA splicing was expected for these six variants as well as for 23 intronic variants in BRCA1 for which the effect on RNA splicing has been published. Out of a total of 174 predictions, 161 (93%) were informative (i.e., the wild-type splice-site was recognized). No false-negative predictions were observed; an effect on RNA splicing was always predicted by these programs. In four cases (2.5%) a false-positive prediction was observed. For DNA diagnostic laboratories, these programs are therefore very useful to select intronic variants that are likely to affect RNA splicing for further analysis.     

The deep intronic c.903+469T>C mutation in the MTRR gene creates an SF2/ASF binding exonic splicing enhancer,which leads to pseudoexon activation and causes the cblE type of homocystinuria

Deep intronic mutations are often ignored as possible causes of human diseases. A deep intronic mutation in the MTRR gene, c.903+469T>C, is the most frequent mutation causing the cblE type of homocystinuria. It is well known to be associated with pre‐mRNA missplicing, resulting in pseudoexon inclusion; however, the pathological mechanism remains unknown. We used minigenes to demonstrate that this mutation is the direct cause of MTRR pseudoexon inclusion, and that the pseudoexon is normally not recognized due to a suboptimal 5′ splice site. Within the pseudoexon we identified an exonic splicing enhancer (ESE), which is activated by the mutation. Cotransfection and siRNA experiments showed that pseudoexon inclusion depends on the cellular amounts of SF2/ASF and in vitro RNA‐binding assays showed dramatically increased SF2/ASF binding to the mutant MTRR ESE. The mutant MTRR ESE sequence is identical to an ESE of the alternatively spliced MST1R proto‐oncogene, which suggests that this ESE could be frequently involved in splicing regulation. Our study conclusively demonstrates that an intronic single nucleotide change is sufficient to cause pseudoexon activation via creation of a functional ESE, which binds a specific splicing factor. We suggest that this mechanism may cause genetic disease much more frequently than previously reported. Hum Mutat 30:1–8, 2010. © 2010 Wiley‐Liss, Inc.