Metab Brain Dis DOI 10.1007/s11011-014-9612-6
RESEARCH ARTICLE
A novel homozygous MCOLN1 double mutant allele leading to TRP channel domain ablation underlies Mucolipidosis IV in an Italian Child Marisol Mirabelli-Badenier & Mariasavina Severino & Barbara Tappino & Domenico Tortora & Francesca Camia & Clelia Zanaboni & Fabia Brera & Enrico Priolo & Andrea Rossi & Roberta Biancheri & Maja Di Rocco & Mirella Filocamo
Received: 10 July 2014 / Accepted: 15 August 2014 # Springer Science+Business Media New York 2014
Abstract Mucolipidosis type IV (MLIV) is a very rare disorder of late endosome/lysosome transport, characterized by neurodevelopmental abnormalities and progressive visual impairment owing to corneal clouding and retinal dystrophy. Greater than 70 % of MLIV patients are of Ashkenazi Jewish ancestry. Here we report a novel MCOLN1double mutant allele [c.395_397delCTG;c.468_474dupTTGG ACC] which introduces a premature stop codon [p.Ala132del; p.Asn159LeufsX27] leading to almost complete abrogation of
Electronic supplementary material The online version of this article (doi:10.1007/s11011-014-9612-6) contains supplementary material, which is available to authorized users. M. Mirabelli-Badenier : F. Camia : F. Brera : R. Biancheri UO Neuropsichiatria Istituto Giannina Gaslini, Genoa, Italy M. Severino : A. Rossi UO Neuroradiologia Istituto Giannina Gaslini, Genoa, Italy B. Tappino : M. Filocamo (*) Centro di Diagnostica Genetica e Biochimica delle Malattie Metaboliche Istituto Giannina Gaslini, Via G. Gaslini 5, 16147 Genoa, Italy e-mail: [email protected] D. Tortora Dipartimento di Radiologia, Università di Chieti, Chieti, Italy C. Zanaboni UO Anestesia e Rianimazione Istituto Giannina Gaslini, Genoa, Italy E. Priolo UO Oculistica Isituto Giannina Gaslini, Genoa, Italy M. Di Rocco Unità Semplice Dipartimentale di Malattie Rare Istituto Giannina Gaslini, Genoa, Italy
the region coding mucolipin-1, a member of the transient receptor potential (TRP) cation channel family. The genomic lesion was identified in homozygous state, in a nonJewish Italian MLIV patient, who also presented abnormal serum gastrin levels. Conventional and advanced MRI sequences, including diffusion tensor imaging and tractography, were used for the assessment of white matter involvement in the patient.
Keywords Lysosomal disorder . Mucolipin 1 double mutant allele . Transient receptor potential cation channel . Brain MRI . Diffusion tensor imaging . Tractography
Abbreviations ADC AJ CC LSD CST DTI DTT FA MCOLN1 MLIV MRI PCR PLIC RT-PCR TRP TRPML1
Apparent diffusion coefficient Ashkenazi Jewish Corpus callosum Lysosomal storage disease Corticospinal tract Diffusion tensor imaging Diffusion tensor tractography Fractional anisotropy Mucolipin 1 gene Mucolipidosis type IV Magnetic resonance imaging Polymerase chain reaction Posterior limb of internal capsule Reverse transcription PCR Transient receptor potential Transient receptor potential channel protein mucolipin-1
Metab Brain Dis
Introduction Mucolipidosis type IV (MLIV, MIM# 252650) is a rare autosomal recessive lysosomal storage disease (LSD) characterized by early infantile onset of neurological symptoms and visual impairment. The majority of patients presents with psychomotor delay, hypotonia gradually progressing to spasticity, bilateral pyramidal tract signs, corneal clouding, strabismus and retinal dystrophy. Achlorhydria and hypergastrinemia are hallmark findings [Schiffmann et al. 2010; Wakabayashi et al. 2011], while dysmorphic features, hepatosplenomegaly, and skeletal deformities are absent. Typical MRI findings include white matter dysmyelination with small corpus callosum and cerebellar atrophy. Only 15 % of patients show neurological deterioration, while retinal dystrophy is usually progressive. Atypical forms with milder clinical phenotype have also been reported, often leading to late diagnosis [Schiffmann et al. 2010]. In contrast to the majority of LSDs, which are characterized by the deficiency of an enzyme, MLIV results from loss-offunction mutations in MCOLN1 gene (MIM# 605248) encoding mucolipin 1 (ML1), also referred to as transient receptor potential channel protein mucolipin-1 (TRPML1), a vesicular Ca2+ release channel belonging to the transient receptor potential (TRP) superfamily [Bargal et al. 2000; Bassi et al. 2000; Cantiello et al. 2005]. ML1 is a membrane protein with six putative transmembrane-spanning domains and both N- and C-termini oriented toward the cytoplasm. It is localized at late endosomes and lysosomes and contains 2 acidic di-leucine consensus motifs located at the N- and C-terminal tails that regulate its trafficking to the late endosomal pathway [Chen et al. 1998; Vergarajauregui and Puertollano 2006]. ML1 alterations result in accumulation of heterogeneous lipids and proteins in cytoplasmic vacuoles derived from lysosomes [Venugopal et al. 2007]. Although little is known about the disease pathogenesis and the associated phenotypic heterogeneity it was suggested that the defective organization of white matter in the brain as well as the reduced maintenance of cells in the retina and optic nerve may result from the inability of cells to compensate for the missing cation channel function [Schiffmann et al. 2010]. Indeed, mucolipin-1 defect may affect the interchange of metabolites between the cytosol and organelles, or it may alter the cell migration that occurs during corpus callosum development which relies on cell-to-cell signaling [Nissenkorn et al. 2001]. The mucolipin 1 function, analyzed in the Mcoln1(−/−) knockout mouse model of mucolipidosis type IV, showed that glial cell activation was increased in brain, and there was evidence of reduced myelination in cerebral and cerebellar white matter tracts [Micsenyi et al. 2009]. Although MLIV is a pan-ethnic disorder, about 70–80 % of patients have Ashkenazi Jewish (AJ) ancestry [Bach et al. 2005; Wakabayashi et al. 2011]. Two founder mutations, a splice mutation (c.416-2A>G) and a partial gene deletion
(c.1_788del), comprise 95 % of the MLIV alleles in this ethnic group [Bargal et al. 2000; Bach et al. 2005]. To date, 28 mutations in AJ and non-AJ patients have been described as isolated case and listed at http://www.hgmd.org [Stenson et al. 2014]. Here we report a novel mutation in the MCOLN1 gene in a non-Jewish Italian MLIV patient studied with both conventional and advanced MRI sequences including diffusion tensor imaging (DTI).
Patient and methods Patient The patient, a 5-year-old male, is the only child of healthy non-consanguineous Italian parents with no known AJ ancestors. Clinical, neuroradiologic, and laboratory investigations were performed. Ethical aspects Following ethical guidelines, the patient’s and parents’ samples were obtained for analysis and storage with the parents’ written informed consent. The consent was sought using a form approved by the local Ethics Committee. Molecular studies Patient and parents DNA samples were extracted from EBV-lymphoblasts and/or blood using QIAmp DNA Blood Mini Kit according to the manufacturer’s protocol (Qiagen). MCOLN1 gene exons and exon–intron boundaries were PCR amplified, using specific primers designed by reference to the genomic sequence (GenBank accession no. NC_000019.1). Total RNA was extracted from patient lymphoblasts using an RNeasy mini kit (QIAGEN, Courtaboeuf, France) and reverse transcribed by means of an Advantage RTfor-PCR kit (BD Biosciences Clontech, Mountain View, CA, USA). RT-PCR was performed using sets of primers designed by reference to the MCOLN1 mRNA sequence (GenBank accession No. NM_ 020533.2). PCR and RT-PCR products were purified and directly sequenced using an ABI 377 DNA automated sequencer with dye terminator cycle sequencing kits (Applied Biosystems, Foster City, CA), and for data analysis, the Sequencing Analysis Software 5.2 (Life Technologies) was used. Mutation nomenclature Nucleotide numbers are derived from cDNA MUCOLN1 sequences (GenBank accession no. NM_ 020533.2). The mutation is described according to current mutation nomenclature guidelines (http://www.hgvs.org/ mutnomen), ascribing the A of the first ATG translational initiation codon as nucleotide +1.
Results Clinical report The child was born at term by dystocic delivery with normal Apgar scores, after an uneventful pregnancy.
Metab Brain Dis
Birth weight was 3.020 Kg, and head circumference was 33.5 cm (10th–25th percentile). After a normal perinatal period, he demonstrated slowing of physical growth, psychomotor developmental delay, and pyramidal signs. The patient was therefore evaluated in the local Hospital, where cerebral palsy was suspected. The Griffiths Mental Development Scale assessed at 19 months of chronological age demonstrated a global mental-age of 9.5 months. Brain MRI performed at 19 months revealed corpus callosum hypoplasia, slight volume loss of the cerebellar folia, mild global reduction of white matter volume, and delayed myelination with involvement of the posterior limbs of the internal capsules (PLIC). Additional focal areas of prominent T2 hyperintensity and T1 hypointensity were noted in the periventricular and deep frontoparietal white matter, associated with dilated perivascular spaces. Finally, diffuse reduction of T2 signal intensity was noted in the thalami and basal ganglia. At 3 years and 3 months the patient was admitted to our Hospital. On physical examination his weight was 13.2 kg (10th percentile), height was 90 cm (5th percentile), and head circumference was 46 cm (
RESEARCH ARTICLE
A novel homozygous MCOLN1 double mutant allele leading to TRP channel domain ablation underlies Mucolipidosis IV in an Italian Child Marisol Mirabelli-Badenier & Mariasavina Severino & Barbara Tappino & Domenico Tortora & Francesca Camia & Clelia Zanaboni & Fabia Brera & Enrico Priolo & Andrea Rossi & Roberta Biancheri & Maja Di Rocco & Mirella Filocamo
Received: 10 July 2014 / Accepted: 15 August 2014 # Springer Science+Business Media New York 2014
Abstract Mucolipidosis type IV (MLIV) is a very rare disorder of late endosome/lysosome transport, characterized by neurodevelopmental abnormalities and progressive visual impairment owing to corneal clouding and retinal dystrophy. Greater than 70 % of MLIV patients are of Ashkenazi Jewish ancestry. Here we report a novel MCOLN1double mutant allele [c.395_397delCTG;c.468_474dupTTGG ACC] which introduces a premature stop codon [p.Ala132del; p.Asn159LeufsX27] leading to almost complete abrogation of
Electronic supplementary material The online version of this article (doi:10.1007/s11011-014-9612-6) contains supplementary material, which is available to authorized users. M. Mirabelli-Badenier : F. Camia : F. Brera : R. Biancheri UO Neuropsichiatria Istituto Giannina Gaslini, Genoa, Italy M. Severino : A. Rossi UO Neuroradiologia Istituto Giannina Gaslini, Genoa, Italy B. Tappino : M. Filocamo (*) Centro di Diagnostica Genetica e Biochimica delle Malattie Metaboliche Istituto Giannina Gaslini, Via G. Gaslini 5, 16147 Genoa, Italy e-mail: [email protected] D. Tortora Dipartimento di Radiologia, Università di Chieti, Chieti, Italy C. Zanaboni UO Anestesia e Rianimazione Istituto Giannina Gaslini, Genoa, Italy E. Priolo UO Oculistica Isituto Giannina Gaslini, Genoa, Italy M. Di Rocco Unità Semplice Dipartimentale di Malattie Rare Istituto Giannina Gaslini, Genoa, Italy
the region coding mucolipin-1, a member of the transient receptor potential (TRP) cation channel family. The genomic lesion was identified in homozygous state, in a nonJewish Italian MLIV patient, who also presented abnormal serum gastrin levels. Conventional and advanced MRI sequences, including diffusion tensor imaging and tractography, were used for the assessment of white matter involvement in the patient.
Keywords Lysosomal disorder . Mucolipin 1 double mutant allele . Transient receptor potential cation channel . Brain MRI . Diffusion tensor imaging . Tractography
Abbreviations ADC AJ CC LSD CST DTI DTT FA MCOLN1 MLIV MRI PCR PLIC RT-PCR TRP TRPML1
Apparent diffusion coefficient Ashkenazi Jewish Corpus callosum Lysosomal storage disease Corticospinal tract Diffusion tensor imaging Diffusion tensor tractography Fractional anisotropy Mucolipin 1 gene Mucolipidosis type IV Magnetic resonance imaging Polymerase chain reaction Posterior limb of internal capsule Reverse transcription PCR Transient receptor potential Transient receptor potential channel protein mucolipin-1
Metab Brain Dis
Introduction Mucolipidosis type IV (MLIV, MIM# 252650) is a rare autosomal recessive lysosomal storage disease (LSD) characterized by early infantile onset of neurological symptoms and visual impairment. The majority of patients presents with psychomotor delay, hypotonia gradually progressing to spasticity, bilateral pyramidal tract signs, corneal clouding, strabismus and retinal dystrophy. Achlorhydria and hypergastrinemia are hallmark findings [Schiffmann et al. 2010; Wakabayashi et al. 2011], while dysmorphic features, hepatosplenomegaly, and skeletal deformities are absent. Typical MRI findings include white matter dysmyelination with small corpus callosum and cerebellar atrophy. Only 15 % of patients show neurological deterioration, while retinal dystrophy is usually progressive. Atypical forms with milder clinical phenotype have also been reported, often leading to late diagnosis [Schiffmann et al. 2010]. In contrast to the majority of LSDs, which are characterized by the deficiency of an enzyme, MLIV results from loss-offunction mutations in MCOLN1 gene (MIM# 605248) encoding mucolipin 1 (ML1), also referred to as transient receptor potential channel protein mucolipin-1 (TRPML1), a vesicular Ca2+ release channel belonging to the transient receptor potential (TRP) superfamily [Bargal et al. 2000; Bassi et al. 2000; Cantiello et al. 2005]. ML1 is a membrane protein with six putative transmembrane-spanning domains and both N- and C-termini oriented toward the cytoplasm. It is localized at late endosomes and lysosomes and contains 2 acidic di-leucine consensus motifs located at the N- and C-terminal tails that regulate its trafficking to the late endosomal pathway [Chen et al. 1998; Vergarajauregui and Puertollano 2006]. ML1 alterations result in accumulation of heterogeneous lipids and proteins in cytoplasmic vacuoles derived from lysosomes [Venugopal et al. 2007]. Although little is known about the disease pathogenesis and the associated phenotypic heterogeneity it was suggested that the defective organization of white matter in the brain as well as the reduced maintenance of cells in the retina and optic nerve may result from the inability of cells to compensate for the missing cation channel function [Schiffmann et al. 2010]. Indeed, mucolipin-1 defect may affect the interchange of metabolites between the cytosol and organelles, or it may alter the cell migration that occurs during corpus callosum development which relies on cell-to-cell signaling [Nissenkorn et al. 2001]. The mucolipin 1 function, analyzed in the Mcoln1(−/−) knockout mouse model of mucolipidosis type IV, showed that glial cell activation was increased in brain, and there was evidence of reduced myelination in cerebral and cerebellar white matter tracts [Micsenyi et al. 2009]. Although MLIV is a pan-ethnic disorder, about 70–80 % of patients have Ashkenazi Jewish (AJ) ancestry [Bach et al. 2005; Wakabayashi et al. 2011]. Two founder mutations, a splice mutation (c.416-2A>G) and a partial gene deletion
(c.1_788del), comprise 95 % of the MLIV alleles in this ethnic group [Bargal et al. 2000; Bach et al. 2005]. To date, 28 mutations in AJ and non-AJ patients have been described as isolated case and listed at http://www.hgmd.org [Stenson et al. 2014]. Here we report a novel mutation in the MCOLN1 gene in a non-Jewish Italian MLIV patient studied with both conventional and advanced MRI sequences including diffusion tensor imaging (DTI).
Patient and methods Patient The patient, a 5-year-old male, is the only child of healthy non-consanguineous Italian parents with no known AJ ancestors. Clinical, neuroradiologic, and laboratory investigations were performed. Ethical aspects Following ethical guidelines, the patient’s and parents’ samples were obtained for analysis and storage with the parents’ written informed consent. The consent was sought using a form approved by the local Ethics Committee. Molecular studies Patient and parents DNA samples were extracted from EBV-lymphoblasts and/or blood using QIAmp DNA Blood Mini Kit according to the manufacturer’s protocol (Qiagen). MCOLN1 gene exons and exon–intron boundaries were PCR amplified, using specific primers designed by reference to the genomic sequence (GenBank accession no. NC_000019.1). Total RNA was extracted from patient lymphoblasts using an RNeasy mini kit (QIAGEN, Courtaboeuf, France) and reverse transcribed by means of an Advantage RTfor-PCR kit (BD Biosciences Clontech, Mountain View, CA, USA). RT-PCR was performed using sets of primers designed by reference to the MCOLN1 mRNA sequence (GenBank accession No. NM_ 020533.2). PCR and RT-PCR products were purified and directly sequenced using an ABI 377 DNA automated sequencer with dye terminator cycle sequencing kits (Applied Biosystems, Foster City, CA), and for data analysis, the Sequencing Analysis Software 5.2 (Life Technologies) was used. Mutation nomenclature Nucleotide numbers are derived from cDNA MUCOLN1 sequences (GenBank accession no. NM_ 020533.2). The mutation is described according to current mutation nomenclature guidelines (http://www.hgvs.org/ mutnomen), ascribing the A of the first ATG translational initiation codon as nucleotide +1.
Results Clinical report The child was born at term by dystocic delivery with normal Apgar scores, after an uneventful pregnancy.
Metab Brain Dis
Birth weight was 3.020 Kg, and head circumference was 33.5 cm (10th–25th percentile). After a normal perinatal period, he demonstrated slowing of physical growth, psychomotor developmental delay, and pyramidal signs. The patient was therefore evaluated in the local Hospital, where cerebral palsy was suspected. The Griffiths Mental Development Scale assessed at 19 months of chronological age demonstrated a global mental-age of 9.5 months. Brain MRI performed at 19 months revealed corpus callosum hypoplasia, slight volume loss of the cerebellar folia, mild global reduction of white matter volume, and delayed myelination with involvement of the posterior limbs of the internal capsules (PLIC). Additional focal areas of prominent T2 hyperintensity and T1 hypointensity were noted in the periventricular and deep frontoparietal white matter, associated with dilated perivascular spaces. Finally, diffuse reduction of T2 signal intensity was noted in the thalami and basal ganglia. At 3 years and 3 months the patient was admitted to our Hospital. On physical examination his weight was 13.2 kg (10th percentile), height was 90 cm (5th percentile), and head circumference was 46 cm (
