Mutations in the Alpha 1,2-Mannosidase Gene, MAN1B1, Cause Autosomal-Recessive Intellectual Disability
2011; Elsevier BV; Volume: 89; Issue: 1 Linguagem: Inglês
10.1016/j.ajhg.2011.06.006
ISSN1537-6605
AutoresMuhammad Rafiq, Andreas W. Kuß, Lucia Puettmann, Abdul Noor, Annapoorani Ramiah, Ghazanfar Ali, Hao Hu, Nadir Ali Kerio, Yong Xiang, Masoud Garshasbi, Muzammil Ahmad Khan, Gisele E. Ishak, Rosanna Weksberg, Reinhard Ullmann, Andreas Tzschach, Kimia Kahrizi, Khalid Mahmood, Farooq Naeem, Muhammad Ayub, Kelley W. Moremen, John B. Vincent, Hans Hilger Ropers, Muhammad Ansar, Hossein Najmabadi,
Tópico(s)Genomics and Rare Diseases
ResumoWe have used genome-wide genotyping to identify an overlapping homozygosity-by-descent locus on chromosome 9q34.3 (MRT15) in four consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability (NS-ARID) and one in which the patients show additional clinical features. Four of the families are from Pakistan, and one is from Iran. Using a combination of next-generation sequencing and Sanger sequencing, we have identified mutations in the gene MAN1B1, encoding a mannosyl oligosaccharide, alpha 1,2-mannosidase. In one Pakistani family, MR43, a homozygous nonsense mutation (RefSeq number NM_016219.3: c.1418G>A [p.Trp473∗]), segregated with intellectual disability and additional dysmorphic features. We also identified the missense mutation c. 1189G>A (p.Glu397Lys; RefSeq number NM_016219.3), which segregates with NS-ARID in three families who come from the same village and probably have shared inheritance. In the Iranian family, the missense mutation c.1000C>T (p.Arg334Cys; RefSeq number NM_016219.3) also segregates with NS-ARID. Both missense mutations are at amino acid residues that are conserved across the animal kingdom, and they either reduce kcat by ∼1300-fold or disrupt stable protein expression in mammalian cells. MAN1B1 is one of the few NS-ARID genes with an elevated mutation frequency in patients with NS-ARID from different populations. We have used genome-wide genotyping to identify an overlapping homozygosity-by-descent locus on chromosome 9q34.3 (MRT15) in four consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability (NS-ARID) and one in which the patients show additional clinical features. Four of the families are from Pakistan, and one is from Iran. Using a combination of next-generation sequencing and Sanger sequencing, we have identified mutations in the gene MAN1B1, encoding a mannosyl oligosaccharide, alpha 1,2-mannosidase. In one Pakistani family, MR43, a homozygous nonsense mutation (RefSeq number NM_016219.3: c.1418G>A [p.Trp473∗]), segregated with intellectual disability and additional dysmorphic features. We also identified the missense mutation c. 1189G>A (p.Glu397Lys; RefSeq number NM_016219.3), which segregates with NS-ARID in three families who come from the same village and probably have shared inheritance. In the Iranian family, the missense mutation c.1000C>T (p.Arg334Cys; RefSeq number NM_016219.3) also segregates with NS-ARID. Both missense mutations are at amino acid residues that are conserved across the animal kingdom, and they either reduce kcat by ∼1300-fold or disrupt stable protein expression in mammalian cells. MAN1B1 is one of the few NS-ARID genes with an elevated mutation frequency in patients with NS-ARID from different populations. Intellectual disability (ID), also called mental retardation (MR), is a devastating neurodevelopmental disorder that has a serious impact on the affected individuals and their families, as well as on health and social services. It is believed to occur with a prevalence of ∼1%–3% within the population,1Roeleveld N. Zielhuis G.A. Gabreëls F. The prevalence of mental retardation: a critical review of recent literature.Dev. Med. Child Neurol. 1997; 39: 125-132Crossref PubMed Scopus (329) Google Scholar, 2Leonard H. Wen X. The epidemiology of mental retardation: Challenges and opportunities in the new millennium.Ment. Retard. Dev. Disabil. Res. Rev. 2002; 8: 117-134Crossref PubMed Scopus (449) Google Scholar and is frequently the result of genetic aberrations. ID can present as the sole clinical feature (nonsyndromic [NS]; see MIM 249500), or it can be present with additional clinical or dysmorphological features (syndromic [S]). Generally speaking, it is much more straightforward to identify the genetic cause of S-ID patients because the comorbid features frequently enable a medical geneticist to narrow down the suspected cause to a mutation in a short list of genes, or perhaps in a single gene. However, for individuals with NS-ID, no secondary clues assist the molecular diagnosis. ID is significantly more frequent in males than in females, and it had been assumed that ∼25% of severe cases were X-linked; however, a recent review suggests that X-linked mutations contribute to no more than 10% of cases,3Ropers H.H. Hamel B.C. X-linked mental retardation.Nat. Rev. Genet. 2005; 6: 46-57Crossref PubMed Scopus (355) Google Scholar and thus it is expected that there will be many more autosomal genes whose mutations cause NS-ID, both dominant and recessive. However, as a result of the high degree of genetic heterogeneity, mutations in only seven genes have been reported to cause NS-ARID, and all of these have been identified on the basis of mapping regions of autozygosity or homozygosity-by-descent (HBD) in multiplex consanguineous families (reviewed in Kaufman et al.4Kaufman L. Ayub M. Vincent J.B. The genetic basis of non-syndromic intellectual disability: A review.J Neurodev Disord. 2010; 2: 182-209Crossref PubMed Scopus (163) Google Scholar). We ascertained three large NS-ARID-affected multiplex consanguineous families from a farming community in the Dera Ghazi Khan district, within Punjab province in Pakistan (MR7, MR8, MR9), one from Sukkur in Sindh province (MR43), and one from Iran (8600060; Figure 1). Appropriate informed consent was obtained for all participants in the study, and institutional research ethics approval was obtained. The description of ascertainment, clinical features, and mapping for families MR7, MR8, and MR9 are given elsewhere5Rafiq M.A. Ansar M. Marshall C.R. Noor A. Shaheen N. Mowjoodi A. Khan M.A. Ali G. Amin-ud-Din M. Feuk L. et al.Mapping of three novel loci for non-syndromic autosomal recessive mental retardation (NS-ARMR) in consanguineous families from Pakistan.Clin. Genet. 2010; 78: 478-483Crossref PubMed Scopus (16) Google Scholar but are summarized in Table 1, Table 2. Two individuals from MR7 (IV:5 and IV:6, age 14 and 13 years, respectively) were assessed and found to have similar developmental history. Both had delayed developmental milestones and began walking at age 4 yr. Speech was also delayed, but they are now both able to speak in sentences, albeit limited to immediate and day-to-day needs. They are toilet trained but require assistance with bathing and dressing. Both have hyperphagia and are overweight. MR7 IV:5 also suffers from asthma. Brain MRI scan for MR7-IV:5 was examined by a radiologist (GI), and showed no evidence of white-matter abnormality within the cerebellum or cerebrum; however, we observed a small prominent perivascular space in the right parietal lobe, as well as cerebellar and cerebral sulci that are mildly prominent but not enough so to constitute volume loss or atrophy, especially given that the ventricles are normal in size (see Figure S1). MR8-IV:1 died in 2009 (from hepatitis C). MR8-IV:2 (age 10 years) has a slightly higher level of functioning than MR7-IV:5 and IV:6 and speaks in sentences. He is toilet trained can dress himself if supervised. He attends school, can read words of one or two syllables, and can count to ten. MR9-II:1 (age 18 years) has a developmental trajectory similar to that of MR7-IV:5 and IV:6. He is able to speak in sentences but is unable to read or write. Inappropriate sexualized behavior is a major cause for concern to the family. He has epileptic seizures, which started at 10 years of age. He has at least one episode per week. Episodes begin with twitching of the facial muscles, then become generalized, at which point he loses consciousness. The seizures have a tonic and then clonic phase and last for several minutes, after which he takes 3 to 4 hr to recover. During this period he is drowsy and disorientated. He frequently becomes incontinent during the fit. Sometimes the family members are able to avert a fit by distracting his attention. MR9-II:3 (age 9 years) has a developmental trajectory similar to that of her sibling, MR9-II:1, but no epilepsy reported to date. Electroencephalographs were not available for MR9-II:1. Verbal and physical aggression is a common feature among affected individuals from MR7 and MR9, but not for individual MR8-IV:2. Mild dysmorphic features were noted for members of families MR7, MR8, and MR9 (see Table 2); however, these cannot be excluded as familial traits, and the families are categorized as nonsyndromic.Table 1Summary of Clinical InformationFamily IDMutation (Nucleotide; Protein)GenderAgeIQSyndromic/NonsyndromicOFC (cm)Height (cm)Obesity (Weight in Kg)EpilepsyMR7- IV:5c.1189G>A; p.Glu397LysM1445–55NS55.5155+ (68)-MR7- IV:6c.1189G>A; p.Glu397LysF1345–55NS54.5150+ (60)-MR8- IV:2c.1189G>A; p.Glu397LysM1050–60NSNKaNK: not known or not measured.NKaNK: not known or not measured.--MR8- IV:1 bNow deceased (from hepatitis C).c.1189G>A; p.Glu397LysFN.K.NSNKaNK: not known or not measured.NKaNK: not known or not measured.--MR9-II-3c.1189G>A; p.Glu397LysF945–55NS51119- (23)-MR9-II-1c.1189G>A; p.Glu397LysM1845–55NSNKaNK: not known or not measured.NKaNK: not known or not measured.-+MR43-VI:4c.1418G>A; p.Trp473∗M955–65S53130- (26.9)+MR43-VI:1c.1418G>A; p.Trp473∗F1655–65NKaNK: not known or not measured.55138- (36)-MR43-VI:5c.1418G>A; p.Trp473∗M855–65S53121- (19)-8600060-V:1c.1000C>T; p.Arg334CysM1159NS53136--8600060-V:2c.1000C>T; p.Arg334CysF2563NS55155--8600060-V:3c.1000C>T; p.Arg334CysF2356NS53.5154--Nucleotide sequence uses NM_016219.3, the reference mRNA sequence for MAN1B1.a NK: not known or not measured.b Now deceased (from hepatitis C). Open table in a new tab Table 2Details of Examination of Photographs of Affected Family MembersFamily IDMutation (Nucleotide; Protein)Physical Features and DysmorphologyEyesHead and EarsFaceHandsFeetMR7- IV:5c.1189G>A; p.Glu397Lysdown-slanting palpebral fissures; thin, sparse eyebrowsmild dolicocephaly; short neckbroad noseMR7- IV:6c.1189G>A; p.Glu397Lysdown-slanting palpebral fissures; thin, sparse eyebrowsmild dolicocephaly; short neckbroad noseMR8- IV:2c.1189G>A; p.Glu397LysNKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).MR8- IV:1bNow deceased (from hepatitis C).c.1189G>A; p.Glu397LysNKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).MR9-II:3c.1189G>A; p.Glu397LysArched, sparse eyebrows;triangular, pointed chinMR9-II:1c.1189G>A; p.Glu397LysArched, sparse eyebrows; long eyelashestriangular, pointed chin flat philtrumNKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).MR43-VI:4c.1418G>A; p.Trp473∗down-slanting palpebral fissures; hyperteloricflat occipitus; low-set earslong face; flattened malar regions; short philtrum; everted lips; broad nasal root; small chinfifth-finger clinodactylypartial second- and third-toe syndactylyMR43-VI:1c.1418G>A; p.Trp473∗NKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).MR43-VI:5c.1418G>A; p.Trp473∗down-slanting palpebral fissures; hyperteloricNKaNK: not known (photographs or clinical information is unavailable to researchers).long face; flattened malar regions; short philtrum; broad nasal root; small chinNKaNK: not known (photographs or clinical information is unavailable to researchers).NKaNK: not known (photographs or clinical information is unavailable to researchers).8600060-V:1c.1000C>T; p.Arg334Cysdown-slanting palpebral fissures; hyperteloric; broad, long eyebrowsflat philtrum; thin upper lip; triangular, pointed chin; prominent nose8600060-V:2c.1000C>T; p.Arg334Cysdown-slanting palpebral fissures; hyperteloric; broad, long eyebrowsmild dolicocephalyflat philtrum; thin upper lip; triangular, pointed chin; prominent nose8600060-V:3c.1000C>T; p.Arg334Cysdown-slanting palpebral fissures; hyperteloric; broad, long eyebrowsmild dolicocephalyflat philtrum; thin upper lip; triangular, pointed chin; prominent noseNucleotide sequence uses the reference mRNA sequence for MAN1B1, NM_016219.3. Photographs will be made available to researchers by the corresponding author, upon signing a nondisclosure agreement.a NK: not known (photographs or clinical information is unavailable to researchers).b Now deceased (from hepatitis C). Open table in a new tab Nucleotide sequence uses NM_016219.3, the reference mRNA sequence for MAN1B1. Nucleotide sequence uses the reference mRNA sequence for MAN1B1, NM_016219.3. Photographs will be made available to researchers by the corresponding author, upon signing a nondisclosure agreement. It was established that MR7, MR8, and MR9 all shared a HBD region on chromosome 9 (9q34.3; between SNPs rs11103117 and rs12238423 [nucleotides 137,667,734–139,811,104 (UCSC March 2006)]; Figure 2), and the locus was assigned as MRT15.5Rafiq M.A. Ansar M. Marshall C.R. Noor A. Shaheen N. Mowjoodi A. Khan M.A. Ali G. Amin-ud-Din M. Feuk L. et al.Mapping of three novel loci for non-syndromic autosomal recessive mental retardation (NS-ARMR) in consanguineous families from Pakistan.Clin. Genet. 2010; 78: 478-483Crossref PubMed Scopus (16) Google Scholar The critical region was 2.14 Mb, and linkage analysis gave LOD score = 4.8.5Rafiq M.A. Ansar M. Marshall C.R. Noor A. Shaheen N. Mowjoodi A. Khan M.A. Ali G. Amin-ud-Din M. Feuk L. et al.Mapping of three novel loci for non-syndromic autosomal recessive mental retardation (NS-ARMR) in consanguineous families from Pakistan.Clin. Genet. 2010; 78: 478-483Crossref PubMed Scopus (16) Google Scholar Although we were unable to establish familial connections in the ancestry of families MR7, MR8, and MR9, comparison of haplotypes suggests a common founder. MR43 has three affected members, VI:1, VI:4, and VI:5 (ages 16, 9, and 8, respectively) with milder, higher functioning intellectual disability. On assessment, VI:4 showed delayed developmental milestones; he sat at 9 months and walked at 2 years. He can talk in sentences and is independent in terms of self care. He can help with chores around the farm. He is sociable and caring but can become verbally and physically aggressive upon provocation and can damage property. On the basis of the assessment, he has mild ID (estimated IQ 55–65). He has epileptic seizures, which started at 6 years of age. These are primary generalized seizures, which start with eye rolling followed by loss of consciousness. Electroencephalographs were not available. His physical appearance shows facial dysmorphic features, including down-slanting palpebral fissures, hypertelorism, a long face, flattened malar regions, a short philtrum, everted lips, a broad nasal root, and a small chin (Table 2). He has fifth-finger clinodactyly. His elder sister (IV:1) and younger brother (IV:5) have a similar developmental trajectory and level of functioning and similar dysmorphic features (IV:1 was not assessed), but no history of epilepsy. Brain computed tomography (CT) scan for an VI:4 was examined by a radiologist (GI) and showed no apparent abnormalities, including no obvious white-matter abnormalities or obvious cerebellar atrophy. For MR43, an HBD region overlapped with that of MRT15 (9q34.3-qter: SNPs rs11103399– rs11137379; nucleotides 136,609,628–140,147,760; Figure 2). The Iranian family, 8600060, had three affected members of ages 11, 23, and 25 years. They had mild to moderate NS-ARID, which was ascertained and mapped as previously described6Kuss A.W. Garshasbi M. Kahrizi K. Tzschach A. Behjati F. Darvish H. Abbasi-Moheb L. Puettmann L. Zecha A. Weissmann R. et al.Autosomal recessive mental retardation: Homozygosity mapping identifies 27 single linkage intervals, at least 14 novel loci and several mutation hotspots.Hum. Genet. 2011; 129: 141-148Crossref PubMed Scopus (40) Google Scholar to 9q34.3 (SNPs rs2031825–rs11137163, 137,166,820–139,653,199; Figure 2), and linkage analysis gave an LOD score = 3.1.6Kuss A.W. Garshasbi M. Kahrizi K. Tzschach A. Behjati F. Darvish H. Abbasi-Moheb L. Puettmann L. Zecha A. Weissmann R. et al.Autosomal recessive mental retardation: Homozygosity mapping identifies 27 single linkage intervals, at least 14 novel loci and several mutation hotspots.Hum. Genet. 2011; 129: 141-148Crossref PubMed Scopus (40) Google Scholar On assessment, the 11-year-old boy (V:1) showed only slight developmental delay. He started to walk at 1.5 years of age. His speech started at age 2.5. He speaks well and can count and use money. He can dress and wash independently and perform day-to-day tasks such as answering the telephone. He has no history of epilepsy, and just mild ID. His sister, V:3, a 23-year-old, has slight developmental delay, but it is more severe than that of her brother, and she shows aggressive behavior. She can dress and wash independently but cannot count or use money. Her speech is less advanced than her brother's. She has no history of epilepsy. Her older sister, V:2, age 25 years, was more delayed than her younger siblings. She began walking at 4 years. She speaks in sentences but cannot count or use money. She has problems with overeating and aggression and has a history of generalized epilepsy. Study of photographs of affected individuals from this family suggested common facial features, including a flat philtrum and prominent nose. However, because photographs of other family members were not available, these cannot be excluded as familial traits, and the family is categorized as nonsyndromic (see Table 1, Table 2). When all the Pakistani and Iranian families are taken into consideration, a combined critical region spanning nucleotides 137,667,734–139,653,199 (∼2.0 Mb) was likely to harbor a gene in which mutations lead to NS-ARID (see Figure 2). This critical region contains 82 RefSeq coding genes (UCSC hg18 March 2006). For the Iranian family, we performed next-generation sequencing by enrichment of the exonic regions within the linkage interval by using a custom Agilent SureSelect array, followed by sequencing with the Illumina Genome Analyzer II platform. Analysis of prospective changes from the critical region indicated DNA variants in just three genes: GPSM1 [MIM 609491] (one base pair deleted at Chr9:139,235,486 (hg19); RefSeq number NM_015597.4; c.1243 delA [p. Thr415Glnfs∗55]), SOHLH1 [MIM 610224] (missense change: c. 916C>A [p.Leu306Met]; RefSeq number NM_001101677.1) and MAN1B1 [MIM 604346] (missense change: c.1000C>T [p.Arg334Cys]; RefSeq number NM_016219.3). However, only the MAN1B1 change segregated in pedigree 8600060. This change, which occurs in exon 7, was not present in 155 Iranians or 191 Germans. For the Pakistani families, a number of genes were selected as candidates for sequencing, but sequence analysis excluded INPP5E [MIM 613037], GRIN1 [MIM 138249], ABCA2 [MIM 600047], CACN1AB, EHTM1, KCNT1 [MIM 608167], CAMSAP1, OLFM1 [MIM 605336], GLT6D1 [MIM 613699], SEC16A [MIM 612854], LHX3 [MIM 600577], ANAPC2 [MIM 606946], PNPLA7 [MIM 612122), and TUBB2C [MIM 602660]. Sequence analysis of GPSM1, SOHLH1, and MAN1B1 was then undertaken. The same 1 bp deletion was observed in GPSM1 in an affected member of MR43, however this did not segregate, and it was present in homozygous form in an unaffected sibling. This deletion occurs within the last exon of GPSM1, but the location is only present within the coding region for one of the four known isoforms of the gene (RefSeq; UCSC Hg18, March 2006) and is either within intron 9 or upstream for the other isoforms. No changes were found in SOHLH1, but a missense mutation in exon 8 and a nonsense mutation in exon 9 were found in MAN1B1 for MR7, MR8, MR9 (NM_016219.3: c. 1189G>A [p.Glu397Lys]), and MR43 (NM_016219.3: c.1418G>A [p.Trp473∗]), respectively. Both the Arg334 and Glu397 residues are conserved across evolution of the animal kingdom (Figure 1). Furthermore, these residues are also conserved across paralogous sequences that are known to have similar enzymatic functions but in different cellular contexts (Figure 3). The c.1189G>A substitution creates a StyI restriction endonuclease cutting site, and the c.1418G>A destroys a BamHI restriction site. We used PCR followed by either StyI or BamH1 digestion to screen a cohort of 252 Pakistani controls, but the mutant alleles were not detected. None of the three MAN1B1 substitutions have been reported in any SNP databases to date and were not present in the 1000 Genomes Version 60.37e. MAN1B1 encodes the protein endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase (ERManI; EC = 3.2.1.113). ERManI and other class 1 α-mannosidases are members of the glycosyl hydrolase family 47 (GH47), are believed to be key enzymes involved in the maturation of N-glycans in the secretory pathway,7Gonzalez D.S. Karaveg K. Vandersall-Nairn A.S. Lal A. Moremen K.W. Identification, expression, and characterization of a cDNA encoding human endoplasmic reticulum mannosidase I, the enzyme that catalyzes the first mannose trimming step in mammalian Asn-linked oligosaccharide biosynthesis.J. Biol. Chem. 1999; 274: 21375-21386Crossref PubMed Scopus (117) Google Scholar, 8Tremblay L.O. Herscovics A. Cloning and expression of a specific human alpha 1,2-mannosidase that trims Man9GlcNAc2 to Man8GlcNAc2 isomer B during N-glycan biosynthesis.Glycobiology. 1999; 9: 1073-1078Crossref PubMed Scopus (93) Google Scholar and contribute to the timing and disposal of misfolded glycoproteins through the endoplasmic-reticulum-associated degradation pathway.9Karaveg K. Siriwardena A. Tempel W. Liu Z.J. Glushka J. Wang B.C. Moremen K.W. Mechanism of class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.J. Biol. Chem. 2005; 280: 16197-16207Crossref PubMed Scopus (97) Google Scholar Enzyme activity involves a direct Ca2+-mediated interaction between the enzyme and substrate,8Tremblay L.O. Herscovics A. Cloning and expression of a specific human alpha 1,2-mannosidase that trims Man9GlcNAc2 to Man8GlcNAc2 isomer B during N-glycan biosynthesis.Glycobiology. 1999; 9: 1073-1078Crossref PubMed Scopus (93) Google Scholar and the protein forms an (αα)7 barrel structure with the Ca2+ ion bound at the core of an active-site pocket in the center of the barrel.9Karaveg K. Siriwardena A. Tempel W. Liu Z.J. Glushka J. Wang B.C. Moremen K.W. Mechanism of class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.J. Biol. Chem. 2005; 280: 16197-16207Crossref PubMed Scopus (97) Google Scholar The Arg334 residue, which is mutated in the Iranian family, is believed to be located at the base of the active-site pocket.9Karaveg K. Siriwardena A. Tempel W. Liu Z.J. Glushka J. Wang B.C. Moremen K.W. Mechanism of class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.J. Biol. Chem. 2005; 280: 16197-16207Crossref PubMed Scopus (97) Google Scholar The Glu397 residue, which is mutated in the Pakistani MR7,MR8, and MR9 families, is also located at this active-site pocket, where it interacts with glycan substrates (see Figure 4; also see Movie S1).9Karaveg K. Siriwardena A. Tempel W. Liu Z.J. Glushka J. Wang B.C. Moremen K.W. Mechanism of class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.J. Biol. Chem. 2005; 280: 16197-16207Crossref PubMed Scopus (97) Google Scholar Arg334 interacts via hydrogen bonding with the 4′ OH on the glycone in the −1 subsite, and the Glu397 residue is involved in hydrogen bonding to the 6′ OH of the +1 mannose residue. Each of these interactions probably makes a minor energetic contribution relative to others in the active site,10Karaveg K. Moremen K.W. Energetics of substrate binding and catalysis by class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.J. Biol. Chem. 2005; 280: 29837-29848Crossref PubMed Scopus (38) Google Scholar but the p.Arg334Cys mutation might alter the electrostatics in the active site in ways that are not easily predicted. The p.Glu397Lys mutation is likely to be disrupting, partially because of the loss of hydrogen bonding to the side chain but also because the longer Lys side chain would probably provide steric hindrance to docking of the +1 residue. Thus, both missense mutations would probably interfere indirectly with the enzyme's ability to bind and recognize the appropriate oligosaccharide substrate. The cDNA encoding the MAN1B1 catalytic domain was used for site-directed mutagenesis, as described previously.9Karaveg K. Siriwardena A. Tempel W. Liu Z.J. Glushka J. Wang B.C. Moremen K.W. Mechanism of class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.J. Biol. Chem. 2005; 280: 16197-16207Crossref PubMed Scopus (97) Google Scholar The constructs were used for transient transfection of HEK293 cells. The wild-type (wt) construct was secreted well and was purified on a Ni2+-NTA column; however, the Cys334 construct was expressed and secreted at ∼20% of that of wt levels, and Lys397 was expressed and secreted at <5% of wt levels. Although the amount of expressed protein for the Lys397 mutant was too low for enzyme activity to be measured, kinetic analysis of the Cys334 mutant indicated a 1327-fold reduction in kcat, a 6.4-fold reduction in Km, and a 205-fold decrease in Kcat/Km (Table 3). It is highly probable that these missense mutations destabilize the protein and thus negatively affect folding and secretion.Table 3Kinetic Analysis of the MAN1B1 Cys334 MutationEnzyme Formkcat (s−1)Km (μM)kcat/Km (s−1/M)kcat/Km kcat/Km(wt)Wild -type1.31 ± 0.24133 ± 498501Cys3340.000987 ± 0.00001520.6 ± 1.347.90.00486The coding region for the catalytic domain of MAN1B19Karaveg K. Siriwardena A. Tempel W. Liu Z.J. Glushka J. Wang B.C. Moremen K.W. Mechanism of class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.J. Biol. Chem. 2005; 280: 16197-16207Crossref PubMed Scopus (97) Google Scholar was subcloned into a modified version of the pXLG vector15Backliwal G. Hildinger M. Chenuet S. Wulhfard S. De Jesus M. Wurm F.M. Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions.Nucleic Acids Res. 2008; 36: e96Crossref PubMed Scopus (152) Google Scholar encoding an NH2-terminal signal sequence from the T. cruzi lysosomal mannosidase,16Vandersall-Nairn A.S. Merkle R.K. O'Brien K. Oeltmann T.N. Moremen K.W. Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi.Glycobiology. 1998; 8: 1183-1194Crossref PubMed Scopus (35) Google Scholar 8× His tag, Strep II tag, and then a TEV protease cleavage site and the MAN1B1 coding region. The construct was transiently transfected into HEK293 suspension culture cells (Freestyle 293-F, Invitrogen) via the TransIT Pro transfection reagent (Mirus). Five days after transfection the conditioned medium was harvested, and the recombinant enzyme was purified by Ni2+-NTA chromatography (QIAGEN) and concentrated by ultrafiltration. Enzyme assays were performed with Man9GlcNAc2-PA as a substrate.9Karaveg K. Siriwardena A. Tempel W. Liu Z.J. Glushka J. Wang B.C. Moremen K.W. Mechanism of class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.J. Biol. Chem. 2005; 280: 16197-16207Crossref PubMed Scopus (97) Google Scholar, 10Karaveg K. Moremen K.W. Energetics of substrate binding and catalysis by class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.J. Biol. Chem. 2005; 280: 29837-29848Crossref PubMed Scopus (38) Google Scholar Substrate concentrations varying from 5 μM to 1 mM. Enzymatic cleavage was monitored by HPLC, and kinetic values were determined with SigmaPlot. Open table in a new tab The coding region for the catalytic domain of MAN1B19Karaveg K. Siriwardena A. Tempel W. Liu Z.J. Glushka J. Wang B.C. Moremen K.W. Mechanism of class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.J. Biol. Chem. 2005; 280: 16197-16207Crossref PubMed Scopus (97) Google Scholar was subcloned into a modified version of the pXLG vector15Backliwal G. Hildinger M. Chenuet S. Wulhfard S. De Jesus M. Wurm F.M. Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions.Nucleic Acids Res. 2008; 36: e96Crossref PubMed Scopus (152) Google Scholar encoding an NH2-terminal signal sequence from the T. cruzi lysosomal mannosidase,16Vandersall-Nairn A.S. Merkle R.K. O'Brien K. Oeltmann T.N. Moremen K.W. Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi.Glycobiology. 1998; 8: 1183-1194Crossref PubMed Scopus (35) Google Scholar 8× His tag, Strep II tag, and then a TEV protease cleavage site and the MAN1B1 coding region. The construct was transiently transfected into HEK293 suspension culture cells (Freestyle 293-F, Invitrogen) via the TransIT Pro transfection reagent (Mirus). Five days after transfection the conditioned medium was harvested, and the recombinant enzyme was purified by Ni2+-NTA chromatography (QIAGEN) and concentrated by ultrafiltration. Enzyme assays were performed with Man9GlcNAc2-PA as a substrate.9Karaveg K. Siriwardena A. Tempel W. Liu Z.J. Glushka J. Wang B.C. Moremen K.W. Mechanism of class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.J. Biol. Chem. 2005; 280: 16197-16207Crossref PubMed Scopus (97) Google Scholar, 10Karaveg K. Moremen K.W. Energetics of substrate binding and catalysis by class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.J. Biol. Chem. 2005; 280: 29837-29848Crossref PubMed Scopus (38) Google Scholar Substrate concentrations varying from 5 μM to 1 mM. Enzymatic cleavage was monitored by HPLC, and kinetic values were determined with SigmaPlot. The Trp473 nonsense mutation identified in family MR43 is thus likely to result in a truncated form of the protein missing many of the residues required for substrate recognition, as well as the Ca2+-binding Thr688 residue. Furthermore, it occurs in exon 9 of a 13 exon gene and therefore most likely leads to nonsense-mediated RNA decay so that only a little mRNA, if any, would survive to protein translation. However, mRNA was not available from any MR43 family members, and as such, we were unable to confirm this experimentally. The lysosomal-storage disease mannosidosis (MIM 248500 for α mannosidosis; MIM 245100 for β mannosidosis) is an autosomal-recessive condition caused by mutations within genes for lysosomal α-mannosidase enzyme MAN2B1 (MIM 609458) and lysosomal β-mannosidase enzyme MANBA (MIM 609489). Alpha-mannosidosis is a rare condition, with an estimated prevalence between 1:500,000 and 1:750,000,11Malm D. Tollersrud O.K. Tranebjaerg L. Månsson J.E. [Alpha-mannosidosis].Tidsskr. Nor. Laegeforen. 1995; 115: 594-597PubMed Google Scholar, 12Meikle P.J. Hopwood J.J. Clague A.E. Carey W.F. Prevalence of lysosomal storage disorders.JAMA. 1999; 281: 249-254Crossref PubMed Scopus (1602) Google Scholar and it varies in severity from prenatal lethal forms to late-onset forms with slow progression. The main features are intellectual disability, hearing loss, myopathy, skeletal abnormalities, and facial dysmorphic features, as well as vulnerability to infections (see GeneReviews in Web Resources). Brain magnetic resonance imaging (MRI) of α-mannosidosis patients typically show white-matter abnormalities as well as cerebellar atrophy and other structural anomalies (see GeneReviews in Web Resources). β -mannosidosis is even rarer, and clinical features include intellectual disability, behavioral abnormalities, and hearing loss; occasionally, there is also facial dysmorphism, skeletal deformation, susceptibility to respiratory infections, and skin lesions.14Bedilu R. Nummy K.A. Cooper A. Wevers R. Smeitink J. Kleijer W.J. Friderici K.H. Variable clinical presentation of lysosomal beta-mannosidosis in patients with null mutations.Mol. Genet. Metab. 2002; 77: 282-290Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar The clinical features of the four families identified with MAN1B1 missense mutations indicate nonsyndromic ARID because only mild dysmorphic features that could not be excluded as family traits were observed, whereas clear dysmorphic features were noted for the family with the nonsense mutation (MR43; see Table 2; photos of patients from MR7 (IV:5, IV:6), MR8 (IV:2), MR9 (II:1, II:3), and MR43 (VI:4, VI:5), as well as individuals V-1, V:2, and V:3 from family 8600060, were examined by R.W.). A more detailed physical examination might be required in order to reveal more subtle anomalies. Thus, we believe that mutation of this endoplasmic reticulum mannosidase gene results in NS-ARID rather than mannosidosis, and thus the disease mechanism is different from disease-causing mutations in the lysosomal α and β mannosidase genes MAN2B1 and MANBA. The identification of mutations causing NS-ARID in MAN1B1 suggests that the ER-associated degradation (ERAD) pathway is a new disease-associated pathway, and this leads us to further speculate that disruption of other ERAD pathway molecules might result in similar clinical presentation. Thus, mutations in MAN1B1 have been found to cause NS-ARID. Furthermore, this is one of the rare cases of NS-ARID genes with increased mutation frequency, and it is tempting to speculate that future investigations into the molecular causes of NS-ARID in larger numbers of patients might demonstrate that disruption of MAN1B1 accounts for more than one percent of the patients. We wish to thank all the members of this family for their willing participation and cooperation with this study and Ines Mueller for technical assistance. J.B.V. is a National Alliance for Research on Schizophrenia and Depression Independent Investigator. This research was supported by grants from the Pakistan Higher Education Commission (NRPU-1118), the Canadian Institutes of Health Research (#MOP-102758), the German Federal Ministry of Education and Research (MRNET 01GS08161-2), the Max Planck Innovation Fund, the Iranian National Science Foundation, and the U.S. National Institutes of Health (GM047533 and DK075322). H.N., H.H.R., K.K., and A.T. are members of the GENCODYS consortium. Download .pdf (.09 MB) Help with pdf files Document S1. One Figure Download .mov (11.31 MB) Help with mov files Movie S1. Animation of the Protein Structure of MAN1B1 Indicating the Location of Missense ResiduesThe structure shows a transparent surface representation of the enzyme in gray and a stick representation of the glycan substrate (thiodisaccharide) in yellow, as in Figure 4. The Ca2+ ion interacting with the 2′ OH and 3′ OH of the glycone residue in the −1 subsite is indicated with orange spacefill. The two amino acids that are mutated, Arg334 and Glu397, in stick form, are shown in blue and are clearly located close to each other; they are believed to form hydrogen bonds (green dotted lines) with the substrate. The molecular animation was generated with MacPymol 1.4 with the PDB file 1X9D.9 The URLs for data presented herein are as follows:GeneReviews, http://www.ncbi.nlm.nih.gov/sites/GeneTests/review?db=GeneTestsOnline Mendelian Inheritance in Man (OMIM), http://www.OMIM.orgUCSC Genome Bioinformatics, http://genome.ucsc.edu1000 Genomes, http://www.1000genomes.orgCLUSTAL 2.1 multiple sequence alignment, http://www.ebi.ac.uk Mutations in the Alpha 1,2-Mannosidase Gene, MAN1B1, Cause Autosomal-Recessive Intellectual DisabilityRafiq et al.The American Journal of Human GeneticsAugust 12, 2011In Brief(The American Journal of Human Genetics 89, 176–182; July 15, 2011) Full-Text PDF Open Archive
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