Identification of predominant GNPTAB gene mutations in Eastern Chinese patients with mucolipidosis II/III and a prenatal diagnosis of mucolipidosis II

Abstract

Mucolipidosis II α/β, mucolipidosis III α/β, and mucolipidosis III γ are autosomal recessive disorders belonging to the family of lysosomal storage disorders caused by deficiency of the UDP-N-acetylglucosamine, a lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) localized in the Golgi apparatus, which is essential for normal processing and packaging of soluble lysosomal enzymes with initiating the first step of tagging lysosomal enzymes with mannose-6-phosphate (M6P). Mucolipidosis II and III are caused by mutations in the GNPTAB and GNPTG genes, and patients with these diseases are characterized by short stature, skeletal abnormalities, and developmental delay. In this study we report 38 patients with mucolipidosis II and III enrolled in Eastern China during the past 8 years. The diagnosis was made based on clinical characteristics and measurement of plasma lysosomal enzyme activity. Sanger sequencing of GNPTAB and/or GNPTG for all patients and real-time quantitative PCR were performed to confirm the diagnosis. In addition, 11 cases of prenatal mucolipidosis II were diagnosed based on measurement of the enzyme activity in amniotic fluid supernatant and genetic testing of cultured amniotic cells. Based on molecular genetic tests, 30 patients were diagnosed with mucolipidosis II α/β, 6 were diagnosed with III α/β and 2 were diagnosed with III γ. Thirty-seven different GNPTAB gene mutations were identified in 29 patients with mucolipidosis II α/β and six patients with III α/β. These mutations included 22 new mutations (p.W44X, p.E279X, p.W416X, p.W463X, p.Q802X, p.Q882X, p.A34P, p.R334P, p.D408N, p.D534N, p.Y997C, p.D1018V, p.L1025S, p.L1033P, c.88_89delAC, c.890_891insT, c.1150_1151insTTA, c.1523delG, c.2473_2474insA, c.2980_2983delGCCT, c.3094delA, and deletion of exon 9). Four new GNPTG gene mutations were identified (c.13delC, p.Y81X, p.G126R and c.609+1delG) in two mucolipidosis III γ patients. Among the 11 cases of prenatal diagnosis, four were mucolipidosis II fetuses, three were heterozygous, and the remaining four were normal fetuses. This study expands the mutation spectrum of the GNPTAB and GNPTG genes and contributes to specific knowledge of mucolipidosis II/III in a population from Eastern China.

Combined in vitro and in silico analyses of missense mutations in GNPTAB provide new insights into the molecular bases of mucolipidosis II and III alpha/beta

“…Whereas the Trp 357 , Leu 358 , Leu 380 , Ile 392 , Ile 395 , Leu 398 , and Phe 420 residues in the CR2 domain may be crucial for intermolecular and/or intramolecular hydrophobic interactions of GlcNAc‐1‐phosphotransferase, the conformation of Pro 355 and Pro 381 may stabilize the molecular structure of the enzyme and, in particular, of the CR2 domain (Dill, ). Most importantly, among conserved residues within the stealth domains, missense mutations leading to amino acid substitutions of Glu 389 , Asp 408 , His 956 , and Arg 986 have been identified in individuals affected by MLII and MLIII alpha/beta, that is, p.Glu389Lys (Velho et al, ), p.Asp408Asn (Wang et al, ), p.His956Tyr (Otomo et al, ), p.His956Arg (Cathey et al, ), p.Arg986Cys (Cobos, Steglich, Santer, Lukacs, & Gal, ; Coutinho et al, ), p.Arg986Gly (Velho et al, ), and p.Arg986His (Mistri et al, ). The clinical description and the molecular analysis of the patient with severe MLII harboring the homozygous mutation p.Glu389Lys (Velho et al, ) have become available and are presented here for the first time (Supporting Information).…”

Disease‐causing missense mutations within the N‐terminal transmembrane domain of GlcNAc‐1‐phosphotransferase impair endoplasmic reticulum translocation or Golgi retention

“…The results presented here are the first to analyze the consequences of missense mutations within the N‐terminal TMD of GlcNAc‐1‐phosphotransferase that were recently identified in patients with MLII/MLIII αβ (Velho et al, 2019; Wang et al, 2019). Our data establish that two of the mutations, namely p.Val27Asp and p.Val28Asp, that cause the more severe form of the disease (MLII), prevent the insertion of the nascent polypeptide chain into the ER lumen and have no detectable enzyme activity.…”

“…Though our study clearly reveals two distinct mechanisms that contribute to the pathogenicity of these mutations, a correlation of our functional data with the clinical outcome of the individual patients is not precise. For instance, both the p.Ala34Pro and p.Glu36Pro mutants are poorly retained in the Golgi and display around 20% GlcNAc‐1‐phosphotransferase activity compared to WT, yet the patient with the p.Ala34Pro mutation was diagnosed with MLII (Wang et al, 2019), whereas the patient with the p.Glu36Pro mutation was diagnosed with MLIII αβ (Velho et al, 2019). In the case of the former, it was reported that the second GNPTAB allele of the patient also had a mutation (c.136C>T; p.Arg46X) which resulted in the codon for arginine changing into a termination codon.…”

“…To date, there are no studies on the role of the N‐terminal transmembrane domain (TMD) of GlcNAc‐1‐phosphotransferase in Golgi localization. Two recent publications have reported the first patient mutations within the N‐terminal TMD of GlcNAc‐1‐phosphotransferase which lead to MLII or MLIII αβ, although the cellular consequences of these mutations were not addressed (Velho et al, 2019; Wang et al, 2019). Here, we report on the functional characterization of these missense mutations, showing that four of the six mutations affect Golgi retention of GlcNAc‐1‐phosphotransferase, whereas the other two prevent insertion into the ER.…”

Placental pathology in an unsuspected case of mucolipidosis type II with secondary hyperparathyroidism in a premature infant

“…MLII is characterized by neonatal onset of coarse facial features, restricted postnatal growth, generalized hypotonia, gingival hypertrophy and multiple skeletal abnormalities including thoracic deformity, clubfeet, long bone deformity, hip dislocation, and cardiac valvular abnormalities [ 1 , [10] , [11] , [12] , [13] , [14] ]. MLIIIα/β is a later-onset form which is typically diagnosed around three years of age and has slower progression [ 1 , [10] , [11] , [12] , [13] , [14] ]. Those with MLIIIα/β typically live into early adulthood, while most patients with MLII die in early childhood, usually with respiratory compromise [ 15 ].…”