Novel compound heterozygous variants in lectin mannose-binding 2-like gene identified in a Chinese autosomal recessive mental retardation-52 (MRT52) patient with phenotype expansion.

To the Editor: Autosomal recessive mental retardation-52 (MRT52, OMIM: 616887) was first reported in 2016 and is characterized by global developmental delay,[1] severe intellectual disability (ID), speech disorder, and seizures, but no dysmorphic features.[1,2] MRT52 is caused by a homozygous mutation in the vesicular integral membrane protein 36 kDa (VIP36)-like protein, which is also named lectin mannose-binding 2-like (LMAN2L) gene (OMIM: 609552).[3] Nufer et al[4] mapped the LMAN2L gene to chromosome 2q11.2. Although the exact LMAN2L function is not clear, evidence suggests that the LMAN2L gene encodes a transmembrane protein (TM) located in the endoplasmic reticulum (ER), where it functions as a cargo receptor in glycoprotein transportation.[4,5] In this study, two novel compound heterozygous variants were identified in the patient. Our study facilitated the clinical diagnosis of the patient and expanded the LMAN2L variants and phenotypic spectrum. The study was approved by the Medical Ethics Committee of West China Second University Hospital of Sichuan University (Approval No. 2017-4-27), and written informed consent was obtained from the parents of the patient, who agreed to join this study. Before pregnancy, a Chinese adult female sought genetic counseling from the Medical Genetics/Prenatal Diagnosis Center of West China Second University Hospital of Sichuan University (Chengdu, China). She reported that her first child (the proband) was a 3-year-old girl diagnosed with general growth retardation, epilepsy, and hearing loss. Physical examination showed that the proband had no obvious dysmorphic features of the hands, feet, and face. Anthropometric examination revealed growth retardation with a 96 cm height, 14 kg body weight, and 46 cm head circumference in the 25 to 50th percentile. The patient showed a general developmental delay. She had difficulty in sitting, standing, walking, speaking, and making social interactions. The patient presented with hypotonia, stereotyped, self-injurious behavior, and physically incapacitated. However, the parents of the proband were healthy and non-consanguineous. The parents had no family history of congenital malformation or genetic abnormalities. Figure 1A shows the pedigree of the family of the patient. The mother requested a definitive genetic diagnosis of the proband to make a targeted prenatal diagnosis for the next pregnancy.Figure 1: (A) Pedigree of the family of the patient. Generations are shown as I–III. Arrow indicates the patient. Squares represent males and circles represent females. (B) EEG of the patient. At 3 years, multifocal sharp or spinous (slow) waves frequently occur in the right occipital and posterior temporal areas during sleep. (C) CMA profile of the patient showing a 339 kb heterozygosity duplication (5039524_5378102) in the chromosome region 9p24.1. (D) Sanger sequencing shows novel LMAN2L compound heterozygous variants inherited from the parents. (a) c.256C > T, p.R86C variant. (b) c.902del, p.F301Sfs∗8 variant. (E) The variant c.256C > T was in exon 2 and c.902del was in exon 8. (F) LMAN2L protein functional domains schematic representation. The protein consists of a signal peptide, an L-type lectin-like domain, a transmembrane domain, and an ER retention signal motif. The variants (c.256C > T, p.R86C and c.902del, p.F301Sfs∗8) were highlighted in box in the protein domains. (G) LMAN2L three-dimensional structure model. (a) Human LMAN2L protein predicted changes caused by the missense mutation p.R86C. (b) Wild-type amino acid at position 86 (arginine), (c) Variant-type amino acid at position 86 (cysteine). (H) Human LMAN2L protein ProtScale analysis of wild type (a) and the variant (p.R86C); (b) which predicts a decrease in overall hydrophobicity. CMA: Chromosomal microarray analysis; EEG: Electroencephalogram; LMAN2L: Lectin mannose-binding 2-like; ER: Endoplasmic reticulum.Trio-based whole exome sequencing (trio-WES) was performed on Genomic DNA (gDNA) of peripheral blood samples from patient and her patients. Sanger sequencing was used to confirm the rare novel compound heterozygous variants. The primers were designed for standard polymerase chain reaction assays using the Primer five software. The forward 5′-GTATGCCACTGCTGAACT-3′ and reverse 5′-TCTGAATGGATTGGTGTTGA-3′, and forward 5′-TCCTGCTGCTCATCTGTA-3′ and reverse 5′-ACAAGTCATCCTTCTTCCAT-3′ primer pairs were used to amplify c.256 and c.902 of the LMAN2L, respectively. Homology modeling of LMAN2L was performed with SWISS MODEL (https://swissmodel.expasy.org/interactive) using the crystal structure of vesicular integral-membrane protein VIP36 (also named as LMAN2L) from Canis lupus familiaris with 62.8% sequence identity with LMAN2L. Protein hydrophilicity was determined using ProtScale (https://web.expasy.org/protscale/). The proband was delivered by cesarean section at 40 weeks of gestation, weighing 3050 g, 49 cm tall with no history of complications. Her medical records showed that she failed the otoacoustic emission for bilaterally hearing screening test at birth. At 2 months, she presented a burst of tonic seizures, including body stiffness and staring eyes that lasted for 10 s. The electroencephalogram (EEG) showed multifocal sharp or spinous (slow) waves that frequently occurred in the right occipital and posterior temporal areas during sleep [Figure 1B]. Kaplan and levetiracetam were used to treat epilepsy, and the curative effect of the drugs was general. The patient suffered seizures twice a month. The motor and intelligence developmental index scores were less than 50 (the average scores: 100), indicating that the developmental progress was delayed, and the limb muscle tension was low. Her patellar and Achilles tendon reflexes were ++, muscle strength was low, and the Babinski sign was negative. Electromyography potentials suggested that the left/right auditory pathway conduction was damaged. However, no abnormalities were found in the cranial magnetic resonance imaging (MRI), and the metabolic screening and electromyography were normal. The patient had a 46, XX karyotype. Chromosomal microarray analysis of the peripheral blood DNA of the patient showed a 339 kb heterozygous duplication (arr[hg19]9p24.1 (5039524_5378102)×3), and a variant of uncertain significance (VUS) was reported [Figure 1C]. The clinical features, neurological findings, and laboratory tests of the patient are summarized in the [Supplementary Table 1, https://links.lww.com/CM9/B262]. A whole-exome sequence (WES) summary of family members of the patient is listed in the [Supplementary Table 2, https://links.lww.com/CM9/B262]. The variants after filtering based on the frequency are listed in the Supplementary Table 3, https://links.lww.com/CM9/B456. According to the bioinformatics analysis, this study revealed the presence of two potential pathogenic variants in the LMAN2L gene (NM_001142292.2) of the proband in a heterozygous form. The variant c.902del was in exon 8 and c.256C > T was in exon 2 [Figure 1E]. Sanger sequencing confirmed the presence of both variants in the proband. DNA analysis of the parents showed that they were heterozygous carriers [Figure 1D]; thus, establishing an in-trans configuration of the variants in the affected children. The LMAN2L protein contains an N-terminal signal peptide, an L-type lectin-like carbohydrate recognition domain, a TM domain, and a C-terminal ER retention signal motif.[3] The variant c.902del was located in the TM domain, which resulted in an adenine deletion at base pair 902 and a premature termination codon introduction at position 309 (p.F301Sfs∗8) [Figure 1F]. The shortened LMAN2L protein lacked the ER retrieval motif, crucial for the membrane receptor recognition on the Golgi apparatus. The variant c.256C>T led to an amino acid substitution (p.R86C), a disease-causing mutation, as predicted by CADD_Phred, SIFT, PolyPhen, MetaSVM, MetaLR, M-CAP, and REVEL tools. The variant was located in the carbohydrate recognition domain [Figure 1F], which conserved cysteines and key residues required for Ca2+, sugar-binding, and the N-glycosylation site. The variant may switch the sugar-binding affinity and impair the function.[1] The LMAN2L homology modeling is shown in Figure 1G(a). LMAN2L wild type and variant type models at amino acid residue 86 are shown (R86 [Figure 1G(b)] and C86 [Figure 1G(c)]). ProtScale analysis confirmed that the corresponding variation in the human LMAN2L protein (p.R86C) caused a decrease in overall hydrophobicity (Figure 1H(a) and H(b)). However, functional validation assays are required to confirm our hypothesis. According to the American College of Medical Genetics and Genomics (ACMG) criteria,[6] the variant c.902del (p.F301Sfs∗8) could be classified as pathogenic (PVS1 + PM2 + PP4), and the variant c.256C > T (p.R86C) could be classified as likely pathogenic (PM2 + PM3 + PP3 + PP4). To date, two families with LMAN2L variations have been reported worldwide.[1,2] The first case reported in 2016 was a large multigenerational consanguineous family from Pakistan, in which seven individuals had global developmental delay, severe ID, and speech disorder. WES identified a recessive LMAN2L homozygous missense mutation (p.R53Q) in the affected family members.[1] However, there was a lack of clinical data such as EEG and MRI results to indicate whether abnormal brain structure or other diseases could be ruled out. In 2019, the second literature described four family members with an autosomal dominant inheritance of a similar phenotype. A frameshift mutation was identified (p.F358Sfs∗16) in LMAN2L by WES. The degree of ID differed among the affected members.[2] Unlike the recessively inherited p.R53Q, the frameshift mutation might operate through a possible dominant-negative mechanism; however, no evidence supports this hypothesis. Consistent with previous reports, the patient in our study presented generalized developmental delay, severe ID, speech disorder, seizures, stereotypical movements, and self-injurious behavior. However, hearing loss and hypotonia, as reported in this patient, were not present in previous patients. Furthermore, our patient showed more severe mental retardation than previously reported, which may be due to congenital hearing impairment. These observations suggested that our patient may have the expanded MRT52 spectrum disorder. According to her medical records, the patient had a normal karyotype. The analysis result of WES indicated that the 339 kb heterozygous duplication at 9p24.1 was inherited from the mother with a normal phenotype. We reclassified the variation according to the ACMG criteria and the Clinical Genome Resource[7]; the result was still VUS. Combined with the trio-WES and Sanger sequencing results, we found two LMAN2L compound heterozygous variants in the patient, and the parents were heterozygous carriers of this gene. Thus, these findings indicated an autosomal recessive pattern of inheritance, similar to the one reported in the family from Pakistan.[1] In conclusion, we report a patient with compound heterozygous variants in the LMAN2L gene and atypical presentation, including hearing loss and dystonia, expanding the recognized phenotype. In addition, the novel variants expand the LMAN2L spectrum. Furthermore, we performed a definite genetic diagnosis of the patient, which is beneficial for subsequent prenatal diagnoses of the family. Funding This research was supported by grants from the National Key Research and Development Program of China (No. 2021YFC1005300) and the Program of Science and Technology Department of Sichuan Province (No. 2022YFS0244). Conflicts of interest None.