MACF1 Mutations Encoding Highly Conserved Zinc-Binding Residues of the GAR Domain Cause Defects in Neuronal Migration and Axon Guidance
2018; Elsevier BV; Volume: 103; Issue: 6 Linguagem: Inglês
10.1016/j.ajhg.2018.10.019
ISSN1537-6605
AutoresWilliam B. Dobyns, Kimberly A. Aldinger, Gisele E. Ishak, Ghayda Mirzaa, Andrew E. Timms, Megan E. Grout, Marjolein H. G. Dremmen, Rachel Schot, Laura Vandervore, Marjon A. van Slegtenhorst, Martina Wilke, Esmee Kasteleijn, Arthur S. Lee, Brenda J. Barry, Katherine R. Chao, Krzysztof Szczałuba, Joyce A. Kobori, Andrea Hanson‐Kahn, Jonathan A. Bernstein, Lucinda Carr, Felice D’Arco, Kaori Miyana, Tetsuya Okazaki, Yoshiaki Saito, Masayuki Sasaki, Soma Das, Marsha M. Wheeler, Michael J. Bamshad, Deborah A. Nickerson, Elizabeth C. Engle, Frans W. Verheijen, Dan Doherty, Grazia M.S. Mancini,
Tópico(s)Cerebrospinal fluid and hydrocephalus
ResumoTo date, mutations in 15 actin- or microtubule-associated genes have been associated with the cortical malformation lissencephaly and variable brainstem hypoplasia. During a multicenter review, we recognized a rare lissencephaly variant with a complex brainstem malformation in three unrelated children. We searched our large brain-malformation databases and found another five children with this malformation (as well as one with a less severe variant), analyzed available whole-exome or -genome sequencing data, and tested ciliogenesis in two affected individuals. The brain malformation comprised posterior predominant lissencephaly and midline crossing defects consisting of absent anterior commissure and a striking W-shaped brainstem malformation caused by small or absent pontine crossing fibers. We discovered heterozygous de novo missense variants or an in-frame deletion involving highly conserved zinc-binding residues within the GAR domain of MACF1 in the first eight subjects. We studied cilium formation and found a higher proportion of mutant cells with short cilia than of control cells with short cilia. A ninth child had similar lissencephaly but only subtle brainstem dysplasia associated with a heterozygous de novo missense variant in the spectrin repeat domain of MACF1. Thus, we report variants of the microtubule-binding GAR domain of MACF1 as the cause of a distinctive and most likely pathognomonic brain malformation. A gain-of-function or dominant-negative mechanism appears likely given that many heterozygous mutations leading to protein truncation are included in the ExAC Browser. However, three de novo variants in MACF1 have been observed in large schizophrenia cohorts. To date, mutations in 15 actin- or microtubule-associated genes have been associated with the cortical malformation lissencephaly and variable brainstem hypoplasia. During a multicenter review, we recognized a rare lissencephaly variant with a complex brainstem malformation in three unrelated children. We searched our large brain-malformation databases and found another five children with this malformation (as well as one with a less severe variant), analyzed available whole-exome or -genome sequencing data, and tested ciliogenesis in two affected individuals. The brain malformation comprised posterior predominant lissencephaly and midline crossing defects consisting of absent anterior commissure and a striking W-shaped brainstem malformation caused by small or absent pontine crossing fibers. We discovered heterozygous de novo missense variants or an in-frame deletion involving highly conserved zinc-binding residues within the GAR domain of MACF1 in the first eight subjects. We studied cilium formation and found a higher proportion of mutant cells with short cilia than of control cells with short cilia. A ninth child had similar lissencephaly but only subtle brainstem dysplasia associated with a heterozygous de novo missense variant in the spectrin repeat domain of MACF1. Thus, we report variants of the microtubule-binding GAR domain of MACF1 as the cause of a distinctive and most likely pathognomonic brain malformation. A gain-of-function or dominant-negative mechanism appears likely given that many heterozygous mutations leading to protein truncation are included in the ExAC Browser. However, three de novo variants in MACF1 have been observed in large schizophrenia cohorts. Microtubules (MTs) and filamentous actin form key structural components of the cytoskeleton, a dynamic intracellular structure that is essential for several basic cell functions, including migration, the formation of cellular processes (including axons and dendrites), axonal guidance, and vesicular trafficking. Mammalian genomes contain two spectraplakins—MACF1 (also known as ACF7) and DST (also known as BPAG1)—that function as actin-MT cross-linkers and essential integrators and modulators of cytoskeletal processes.1Goryunov D. Liem R.K. Microtubule-actin cross-linking factor 1: domains, interaction partners, and tissue-specific functions.Methods Enzymol. 2016; 569: 331-353Crossref PubMed Scopus (18) Google Scholar, 2Moffat J.J. Ka M. Jung E.M. Smith A.L. Kim W.Y. The role of MACF1 in nervous system development and maintenance.Semin. Cell Dev. Biol. 2017; 69: 9-17Crossref PubMed Scopus (19) Google Scholar, 3Lane T.R. Fuchs E. Slep K.C. Structure of the ACF7 EF-hand-GAR module and delineation of microtubule binding determinants.Structure. 2017; 25: 1130-1138.e6Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar MACF1 is a large gene that expresses many isoforms, several of which are brain specific, through alternative splicing. The N terminus of the major isoforms contains two calponin-homology (CH) domains that bind actin, a plakin domain, and a long spectrin-repeat rod domain that confers flexibility. The C terminus of all isoforms functions as a MT binding domain and contains two calcium-binding EF-hand domains, a zinc-binding GAR (growth-arrest specific 2 or Gas2-related) domain, a positively charged Gly-Ser-Arg (GSR) region, and an EB1-binding SxIP domain.1Goryunov D. Liem R.K. Microtubule-actin cross-linking factor 1: domains, interaction partners, and tissue-specific functions.Methods Enzymol. 2016; 569: 331-353Crossref PubMed Scopus (18) Google Scholar, 2Moffat J.J. Ka M. Jung E.M. Smith A.L. Kim W.Y. The role of MACF1 in nervous system development and maintenance.Semin. Cell Dev. Biol. 2017; 69: 9-17Crossref PubMed Scopus (19) Google Scholar, 3Lane T.R. Fuchs E. Slep K.C. Structure of the ACF7 EF-hand-GAR module and delineation of microtubule binding determinants.Structure. 2017; 25: 1130-1138.e6Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar This work began after we recognized a complex brain malformation consisting of lissencephaly (LIS), a rare brainstem malformation with deficient midline crossing, and multiple developmental deficits including severe intellectual disability and epilepsy in three unrelated children during intergroup reviews. We next searched our large brain-malformation databases (∼3,300 subjects) and identified the same pattern in another five children for a total of eight, including monozygotic twin sisters and one previously reported Japanese girl (Figures 1, 2, S1, and S2).4Irahara K. Saito Y. Sugai K. Nakagawa E. Saito T. Komaki H. Nakata Y. Sato N. Baba K. Yamamoto T. et al.Pontine malformation, undecussated pyramidal tracts, and regional polymicrogyria: a new syndrome.Pediatr. Neurol. 2014; 50: 384-388Abstract Full Text Full Text PDF PubMed Scopus (6) Google ScholarFigure 2Brain MRI in Subjects with MACF1 Zinc-Binding-Pocket Mutations: Additional FeaturesShow full captionAxial (left two columns), coronal (third column), and reconstructed coronal (far-right column) images are shown for (the same as in Figure 1) subjects LR14-088 (A–D), LR17-434 (E–H), LR16-306 (I–L), and LR17-450 (M–P). MACF1 mutations are again shown in the far-left column. The low midbrain or isthmus appears small and narrow with variably prominent superior cerebellar peduncles that are smaller than seen in the molar-tooth malformation associated with Joubert syndrome (especially E, I, and M). The anterior commissures are thin (arrows in B, F, J, and N). The hippocampi are small and dysplastic (C, G, K, and O). The pyramidal tracts are easy to follow given the paucity of transverse pontine crossing fibers; a few are seen in the top two images (asterisks over the right pyramidal tracts in D and H), but none are seen in the lower images (asterisks in L and P).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Axial (left two columns), coronal (third column), and reconstructed coronal (far-right column) images are shown for (the same as in Figure 1) subjects LR14-088 (A–D), LR17-434 (E–H), LR16-306 (I–L), and LR17-450 (M–P). MACF1 mutations are again shown in the far-left column. The low midbrain or isthmus appears small and narrow with variably prominent superior cerebellar peduncles that are smaller than seen in the molar-tooth malformation associated with Joubert syndrome (especially E, I, and M). The anterior commissures are thin (arrows in B, F, J, and N). The hippocampi are small and dysplastic (C, G, K, and O). The pyramidal tracts are easy to follow given the paucity of transverse pontine crossing fibers; a few are seen in the top two images (asterisks over the right pyramidal tracts in D and H), but none are seen in the lower images (asterisks in L and P). According to our revised classification system for LIS,5Di Donato N. Chiari S. Mirzaa G.M. Aldinger K. Parrini E. Olds C. Barkovich A.J. Guerrini R. Dobyns W.B. Lissencephaly: expanded imaging and clinical classification.Am. J. Med. Genet. A. 2017; 173: 1473-1488Crossref PubMed Scopus (73) Google Scholar the cortical malformation consists of diffuse pachygyria with thick cortex and a mild posterior gradient more severe than the anterior gradient; it varied from "thin" LIS with mildly thick cortex (7–10 mm) in six of eight individuals to classic LIS with very thick cortex (10–15 mm) in two of eight individuals. We found thin anterior commissures and mildly dysplastic hippocampi in all eight individuals and mildly thin corpus callosum in six of eight subjects. The brainstem malformation consisted of severe dorso-ventral narrowing of the brainstem mostly in the pons and small ventral prominences on mid-sagittal images (Table 1 and Figures 1 and S1). On axial images, the lower midbrain and entire pons and medulla were narrow on the dorso-ventral axis with a prominent ventral midline cleft but were also very wide, resembling a thick "W" (or wide bird wings when turned upside down), on the right-left axis (see Figures 1B, 1F, and 1J). The base of the pons was small (4–5 mm) in three individuals, tiny (2–3 mm) in three individuals, and absent in two of eight individuals. Cranial nerves could be seen exiting from the ventral surface of both medial and lateral aspects of the dysplastic brainstem. Coronal 3D image reconstructions along the ventral brainstem showed absent or very sparse transverse pontine crossing fibers, uncovering the pyramidal tracts (Figures 2 and S2). The cerebellar vermis was moderately small in two individuals, mildly small in five individuals, and normal in one of eight individuals, whereas the cerebellar hemispheres were mildly small in seven individuals and normal in (the same) one individual. Reconstructed diffusion tensor imaging sequences in one boy (LR17-434) showed small superior cerebellar peduncles and absent anterior and posterior transverse pontine crossing fibers (Figure 3). Similar changes were shown in the previously reported Japanese child.4Irahara K. Saito Y. Sugai K. Nakagawa E. Saito T. Komaki H. Nakata Y. Sato N. Baba K. Yamamoto T. et al.Pontine malformation, undecussated pyramidal tracts, and regional polymicrogyria: a new syndrome.Pediatr. Neurol. 2014; 50: 384-388Abstract Full Text Full Text PDF PubMed Scopus (6) Google ScholarTable 1Brain Imaging Features of Individuals with MACF1 MutationsSubject IDLIS with Brainstem Hypoplasia and DysplasiaLIS OnlyLR14-088LR17-434LR16-306LR17-450LR04-067a1LR04-067a2LR18-077LR18-0704Irahara K. Saito Y. Sugai K. Nakagawa E. Saito T. Komaki H. Nakata Y. Sato N. Baba K. Yamamoto T. et al.Pontine malformation, undecussated pyramidal tracts, and regional polymicrogyria: a new syndrome.Pediatr. Neurol. 2014; 50: 384-388Abstract Full Text Full Text PDF PubMed Scopus (6) Google ScholarLR16-412Protein VariantsGenBank: NM_012090.5aGenBank: NM_012090.5 and transcript MACF1-203 (Ensembl: ENST00000361689.6).p.Cys5177Phep.Cys5177Phep.Asp5228Tyrp.Asp5228Tyrp.Cys5230Phep.Cys5230Phep.Cys5230Glydeletionp.Gly4706ArgMACF1-204bTranscript MACF1-204 (Ensembl: ENST00000372915.7).p.Cys7135Phep.Cys7135Phep.Asp7186Tyrp.Asp7186Tyrp.Cys7188Phep.Cys7188Phep.Cys7188Glydeletionp.Gly6664ArgCerebral HemispheresLIS severityPGY diffusePGY diffusePGY diffusePGY diffusePGY diffusePGY diffusePGY diffusePGY diffusePGY diffuseLIS gradientP > AP > AP > AP > AA = PA = PP > AP > AP > ALIS cortical thicknessthin (4–7 mm)thin (4–7 mm)thick (10–15 mm)thick (10–15 mm)thin (4–7 mm)thin (4–7 mm)thin (4–7 mm)thin (4–7 mm)thin (4–7 mm)Hippocampal dysplasiayesyesyesyesyesyesyessubtlesubtleThin white matteryesyesyesyesyesyesyesyesyesCommissuresAnterior commissurethinthinthincLow-resolution scan.thinborderline thinborderline thinthinthinnormalCorpus callosumthin diffusethin diffusethin diffusedSevere hypoplasia of splenium.thin diffusedSevere hypoplasia of splenium.normalnormalthin mildnormalthin mildHippocampal commissurenormalnormalnormalnormalnormalnormalnormalnormalnormalOptic chiasmnormalnormalnormalnormalnormalnormalnormalnormalnormalBrainstemThick tectumeFused colliculi.nononoyesnononononoSevere narrowing of ponsyesyesyesyesyesyesyesyesnoWide pons and medullayesyesyesyesyesyesyesyessubtleBase of ponstinytinyabsentcLow-resolution scan.absentsmallsmallsmalltinynoPontine hypoplasiaseveresevere (DTI)severeseveremoderate to severemoderate to severemoderate to severeseverenoCerebellumVermis hypoplasiamoderatemildmildmildmildmildmoderatenonormalHemisphere hypoplasiamoderatemildmildmildmildfmildfRight smaller than left.mildnonormalFoliar dysplasiayesyesyesyesyesyesyesyesnoAbbreviations are as follows: A = P, anterior gradient same as posterior gradient; DTI, diffusion tensor imaging; LIS, lissencephaly; P > A, posterior gradient more severe than anterior gradient; PGY, pachygyria.a GenBank: NM_012090.5 and transcript MACF1-203 (Ensembl: ENST00000361689.6).b Transcript MACF1-204 (Ensembl: ENST00000372915.7).c Low-resolution scan.d Severe hypoplasia of splenium.e Fused colliculi.f Right smaller than left. Open table in a new tab Abbreviations are as follows: A = P, anterior gradient same as posterior gradient; DTI, diffusion tensor imaging; LIS, lissencephaly; P > A, posterior gradient more severe than anterior gradient; PGY, pachygyria. All eight children had global developmental delay, severe intellectual disability, axial hypotonia (with limb spasticity in three of eight), and seizures (Table 2). The twin girls were non-verbal and had the most severe intellectual disability, as well as short stature and microcephaly (3–5 standard deviations below the mean), although their pregnancy had been complicated only by gestational diabetes and premature birth at 37 weeks of gestation. One girl spoke more than 40 single words, and another used an assistive language device; the remainder used few or no words. Growth was normal except for in the twins. Seizure types included infantile spasms in two individuals (which resolved after steroid treatment in one), myoclonic seizures in two individuals, and mixed partial and generalized seizures in the remaining four children. Involuntary movements were seen in three of eight individuals, and stereotypies were observed in two others. Cranial-nerve deficits consisted of dysphagia in four individuals, asymmetric facial movements in one individual, strabismus in three individuals, and limited ocular adduction in the previously reported child who improved by 5 years.4Irahara K. Saito Y. Sugai K. Nakagawa E. Saito T. Komaki H. Nakata Y. Sato N. Baba K. Yamamoto T. et al.Pontine malformation, undecussated pyramidal tracts, and regional polymicrogyria: a new syndrome.Pediatr. Neurol. 2014; 50: 384-388Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar Cortical visual impairment was diagnosed in two children, and left optic-nerve hypoplasia was diagnosed in one, whereas hearing was normal in all. One individual had poor temperature regulation plus a neurogenic bladder and bowel attributed to spinal-cord dysfunction without structural abnormality. No consistent dysmorphic facial features were observed, and no other malformations were found, except for a ventricular septal defect that closed spontaneously in one child.Table 2Clinical Features and Protein Variants in Individuals with MACF1 MutationsSubject IDLIS with Brainstem Hypoplasia and DysplasiaLIS OnlyLR14-088LR17-434LR16-306LR17-450LR04-067a1LR04-067a2LR18-077LR18-0704Irahara K. Saito Y. Sugai K. Nakagawa E. Saito T. Komaki H. Nakata Y. Sato N. Baba K. Yamamoto T. et al.Pontine malformation, undecussated pyramidal tracts, and regional polymicrogyria: a new syndrome.Pediatr. Neurol. 2014; 50: 384-388Abstract Full Text Full Text PDF PubMed Scopus (6) Google ScholarLR16-412Protein VariantsGenBank: NM_012090.5,aGenBank: NM_012090.5 and transcript MACF1-203 (Ensembl: ENST00000361689.6).p.Cys5177Phep.Cys5177Phep.Asp5228Tyrp.Asp5228Tyrp.Cys5230Phep.Cys5230Phep.Cys5230GlydeletionbDeletion of exons 58–89 (p.Ala3540_Arg5192; GenBank: NM_012090.5) or exons 62–93 (p.Ala5498_Arg7150; MACF1-204).p.Gly4706ArgMACF1-204cTranscript MACF1-204 (Ensembl: ENST00000372915.7).p.Cys7135Phep.Cys7135Phep.Asp7186Tyrp.Asp7186Tyrp.Cys7188Phep.Cys7188Phep.Cys7188GlydeletionbDeletion of exons 58–89 (p.Ala3540_Arg5192; GenBank: NM_012090.5) or exons 62–93 (p.Ala5498_Arg7150; MACF1-204).p.Gly6664ArgIdentity DataSexfemalemalefemalemalefemalefemalemalefemalefemaleEthnicityC (Indian)C (Dutch)C (USA)C (Polish)C (Hispanic)C (Hispanic)C (Syrian)A (Japanese)C and A (Filipino)Geographic originMumbaiRotterdamBay Area, CAWarsawSan Jose, CASan Jose, CALondonSaitamaSeattle, WAGrowth DataOFC at birth (SD)36 cm (+1.1)NDND31 cm (−1)dOFC at 36 weeks of gestation.32.5 cm (−1.5)NDND33.5 cm (+0.4)NDAge at last exam5 years7.5 years5.5 years7 years16 years16 years7 years2.5 years5 yearsWeight (SD)13.7 kg (−1.7)28 kg (+1)13.5 kg (+1.7)14 kg (−3)35.2 kg (−3)29.0 kg (−4)22 kg (−0.4)10.0 kg (−1.6)20.4 kg (+0.7)Height (SD)105 cm (−0.5)128 cm (0)103 cm (−1)116 cm (−1)150 cm (−2)137 cm (−4)50 cm (−2)82.5 cm (−1.6)107.9 cm (−0.1)OFC (SD)50.5 cm (0)53 cm (+0.7)45.5 cm (−1)47 cm (+0.5)eOFC at 1 year.49.5 cm (−4)47.7 cm (−5)ND47.2 cm (−0.4)51.3 cm (0)Development and Neurological DataDevelopmental delayglobalglobalglobalglobalglobalglobalglobalglobalmildHypotoniayesyesyesyesyesyesyesnonoSpasticitynonononoyesyesyes (legs)nonoSitting (age)yes (1 year)nononoyes (1 year)fSits with support only.noyes (1.5 years)yes (1 year)yes (7 months)Walking (age)yes (3 years)nononoyes (5 years)noyes (7 years)gFew steps only.yes (3 years)hWalks with support at 3 years.yes (1.5 years)Language (age)>40 wordsiAlso with several word combinations.10 syllablesnonejUses Picture Exchange Communication System (PECS).none3 wordskWords later lost.3 wordskWords later lost.nonenoneyes (1.5 years)Intellectual disabilitysevereseveresevereseveresevereseveresevereseveresevereSeizure onset5 months6 months3 months3 months7 months5 months6 months5 years4 years, 3 monthsSeizure typesISSISS, LGSSE, LGSMYOFSIA, LGSFSIA, FTCS, GTCSprobable GTCSMYO, GTCSFSIA, GTCSDyskinesialMixed abnormal movements with combinations of chorea, athetosis, dystonia, and (in the twins) ballismus.hand flappingmixedNDnomixedmixedmixedhand wavingnoVision abnormalitiesCVI, left ONHnoCVInonormalnormalNDNDnormalEye movementsabnormal NOSabnormal NOSnormalnonystagmus horizontalNDslow trackingabduction limitedmOcular abduction limited to half normal excursion at 2.5 years but improved to normal abduction by 5 years.normalStrabismusleft exotropiaexotropiaNDnoNDesotropiaNDNDnoFeeding abnormalitynoneimpaired, GTimpaired, GTnoNDNDnonenonenoOtherFeatureVSD closednonenonenonenonenonedysmorphicnHypertelorism, low nasal bridge, epicanthal folds, and low-set ears.nonenoneAbbreviations are as follows: A, Asian; C, Caucasian; CVI, cortical visual impairment; FSIA, focal seizure with impaired awareness; FTCS, focal tonic-clonic seizure; GT, gastrostomy tube; GTCS, generalized tonic-clonic seizure; ISS, infantile spasm; LGS, Lennox-Gastaut epilepsy syndrome with atonic, tonic, tonic-clonic, and myoclonic seizures; MYO, myoclonic seizure; ND, no data available; NOS, not otherwise specified; OFC, occipitofrontal circumference; ONH, optic-nerve hypoplasia; SD, standard deviation; VSD, ventriculoseptal defect.a GenBank: NM_012090.5 and transcript MACF1-203 (Ensembl: ENST00000361689.6).b Deletion of exons 58–89 (p.Ala3540_Arg5192; GenBank: NM_012090.5) or exons 62–93 (p.Ala5498_Arg7150; MACF1-204).c Transcript MACF1-204 (Ensembl: ENST00000372915.7).d OFC at 36 weeks of gestation.e OFC at 1 year.f Sits with support only.g Few steps only.h Walks with support at 3 years.i Also with several word combinations.j Uses Picture Exchange Communication System (PECS).k Words later lost.l Mixed abnormal movements with combinations of chorea, athetosis, dystonia, and (in the twins) ballismus.m Ocular abduction limited to half normal excursion at 2.5 years but improved to normal abduction by 5 years.n Hypertelorism, low nasal bridge, epicanthal folds, and low-set ears. Open table in a new tab Abbreviations are as follows: A, Asian; C, Caucasian; CVI, cortical visual impairment; FSIA, focal seizure with impaired awareness; FTCS, focal tonic-clonic seizure; GT, gastrostomy tube; GTCS, generalized tonic-clonic seizure; ISS, infantile spasm; LGS, Lennox-Gastaut epilepsy syndrome with atonic, tonic, tonic-clonic, and myoclonic seizures; MYO, myoclonic seizure; ND, no data available; NOS, not otherwise specified; OFC, occipitofrontal circumference; ONH, optic-nerve hypoplasia; SD, standard deviation; VSD, ventriculoseptal defect. The nine subjects were ascertained from multiple centers and enrolled in research after written informed consent was provided under protocols approved by institutional review boards at Seattle Children's Hospital, the University of Washington, Boston Children's Hospital, and Erasmus University Medical Center. Blood or tissue samples were obtained and genomic DNA was extracted according to standard protocols. Whole-exome sequencing was performed in six trios and one proband in seven different laboratories with sequencing parameters that varied modestly across centers and center-specific pipelines (Table S1 and Supplemental Material and Methods). These data were supplemented by whole-genome sequencing in two trios and Sanger sequencing of small critical regions in two probands (the second twin and LR18-077). When no coding changes were found, we looked for small deletions and duplications by manual inspection of read depth in regions where mutations were found in other affected individuals. We found recurrent, heterozygous de novo missense variants involving three of the four highly conserved zinc-binding residues in the GAR domain of MACF1 in seven of eight children (Tables 1, 2, and S2 and Figure 4A). All variants were predicted to be deleterious: all CADD scores (28–34) were well above our standard cutoff of 15, and all PolyPhen-2 scores (0.995–0.999) were rated as probably damaging (Table S2). None of these variants were found in gnomAD. No sequence variants of MACF1 were detected in the Japanese girl,4Irahara K. Saito Y. Sugai K. Nakagawa E. Saito T. Komaki H. Nakata Y. Sato N. Baba K. Yamamoto T. et al.Pontine malformation, undecussated pyramidal tracts, and regional polymicrogyria: a new syndrome.Pediatr. Neurol. 2014; 50: 384-388Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar but manual inspection of read depth and analysis of exome sequence data with the GATK4 GenomeCNVCaller and of genome data with the Manta SV caller detected a 39.6 kb deletion of exons 62–93 (transcript MACF1-204), causing a predicted in-frame deletion from Ala5498 through Arg7150 (Table S2). This region contains the last ten spectrin repeats, both EF-hand domains, and the first two zinc-binding residues: Cys7133 and Cys7135 (Figures 4A and S3). We designed primers flanking the predicted breakpoints to generate a 1.1 kb PCR product and defined breakpoint-flanking sequences that in the normal genome map to intronic sequences 39.6 kb apart: 5′-CCGGGGATTCTGTGTCATCTTAATA-3′–breakpoint–5′-TACAGTAAGCTGAGATCACACTGT-3′. The GAR domain mediates MT binding and consists of a novel zinc-binding α-β fold composed of a five-stranded anti-parallel β sheet (β1–β5) flanked by N-terminal (α1) and C-terminal (α2) α helices that pack against the β sheet to form an α/β sandwich.3Lane T.R. Fuchs E. Slep K.C. Structure of the ACF7 EF-hand-GAR module and delineation of microtubule binding determinants.Structure. 2017; 25: 1130-1138.e6Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar The α1-β1 loop and the C-terminal loop flanking α2 contain four highly conserved residues (Cys7133, Cys7135, Asp7186, and Cys7188 according to Ensembl sequence MACF1-204) that coordinate the bound zinc ion.3Lane T.R. Fuchs E. Slep K.C. Structure of the ACF7 EF-hand-GAR module and delineation of microtubule binding determinants.Structure. 2017; 25: 1130-1138.e6Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar Our first seven subjects (including both twins) had recurrent missense variants involving three of the four zinc-binding residues. These are shown with both the standard NCBI sequence GenBank: NM_021090.5 and the longer Ensembl sequence MACF1-204 (Table S2). Hereafter, we refer only to the MACF1-204 transcript, which has been used for most functional studies.3Lane T.R. Fuchs E. Slep K.C. Structure of the ACF7 EF-hand-GAR module and delineation of microtubule binding determinants.Structure. 2017; 25: 1130-1138.e6Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar Although the phenotypes were remarkably similar, we found several subtle differences. The two with p.Cys7135Phe had more severe hypoplasia of the anterior commissure, and the two with p.Asp7186Tyr had more severe LIS. The three with p.Cys7188Phe or p.Cys7188Gly and the child with the intragenic deletion had larger (3–4 mm) nodules representing residual pontine crossing fibers on the ventral surface of the pons; these were not seen or barely seen in the other four individuals (Table 1). We used the 3D modeling tool PyMOL to predict the effect of missense variants on protein structure of the GAR domain, which was previously resolved by molecular crystallography (Supplemental Material and Methods).3Lane T.R. Fuchs E. Slep K.C. Structure of the ACF7 EF-hand-GAR module and delineation of microtubule binding determinants.Structure. 2017; 25: 1130-1138.e6Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar This region contains a sandwich structure and a divalent zinc-binding pocket formed by four highly conserved amino acids (three cysteines and one aspartate) that are invariant in spectraplakins and essential for MT binding. Substitutions involving these residues were predicted to significantly alter configuration of the zinc-binding pocket (Figure 4B), which supports experimental data showing compromised domain function when one of the cysteine residues is replaced by a serine and strongly supports pathogenicity of the protein variants, which most likely abrogate MT binding.3Lane T.R. Fuchs E. Slep K.C. Structure of the ACF7 EF-hand-GAR module and delineation of microtubule binding determinants.Structure. 2017; 25: 1130-1138.e6Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar Studies using several cre drivers in mouse Macf1 knockouts demonstrated that loss of Macf1 in the retina, ependymal and cochlear sensory epilthelia, and mouse embryonic fibroblasts led to failure of cells to build cilia and disrupted apicobasal polarity in the retina, whereas heterozygous mutants had normal cilia.6May-Simera H.L. Gumerson J.D. Gao C. Campos M. Cologna S.M. Beyer T. Boldt K. Kaya K.D. Patel N. Kretschmer F. et al.Loss of MACF1 abolishes ciliogenesis and disrupts apicobasal polarity establishment in the retina.Cell Rep. 2016; 17: 1399-1413Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar We hypothesized that heterozygous variants in the GAR domain, if pathogenic, would also disrupt cilia formation, a key cellular function that can be tested in cells exiting mitosis after serum starvation.7Reiter J.F. Leroux M.R. Genes and molecular pathways underpinning ciliopathies.Nat. Rev. Mol. Cell Biol. 2017; 18: 533-547Crossref PubMed Scopus (699) Google Scholar We therefore tested cilium formation in skin fibroblasts from two subjects with MACF1 variants and compared it with that of mutant fibroblasts with known defects in cilium number and length (CEP290) or length alone (RTTN).8Kheradmand Kia S. Verbeek E. Engelen E. Schot R. Poot R.A. de Coo I.F. Lequin M.H. Poulton C.J. Pourfarzad F. Grosveld F.G. et al.RTTN mutations link primary cilia function to organization of the human cerebral cortex.Am. J. Hum. Genet. 2012; 91: 533-540Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Fibroblast lines from two control and five affected individuals with known mutations (two in MACF1, two in RTTN, and one in CEP290) were processed in parallel in duplicate or quadruplicate as previously described (Supplemental Material and Methods). In brief, cells were grown until they were 80% confluent, serum starved for 48 hr for the induction of cilium formation, fixed with methanol, incubated overnight with primary antibodies—mouse monoclonal anti-human acetylated tubulin (Sigma Aldrich T7451) and rabbit polyclonal anti-human gamma tubulin (Sigma Aldrich T3320)—and then incubated with secondary fluorescent antibodies for 1 hr. Cells with basal bodies (γ-tubulin positive) were scored for cilium length, defined as normal (>3 μm) or short (<3 μm), in four quadrants for each coverslip by one individual. Values indicate the average length ± SEM. Under these conditions, 50%–80% of cells grew cilia according to data from 100 experiments using multiple control fibroblast lines. In contrast to the mouse homozygous mutants, human fibroblasts carrying MACF1 mutations made
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