Mild Neurodevelopmental Disorder Due to SHMT2 Mutations: Expanding the Phenotypic Spectrum

doi:10.21203/rs.3.rs-5035114/v1

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Mild Neurodevelopmental Disorder Due to SHMT2 Mutations: Expanding the Phenotypic Spectrum

https://doi.org/10.21203/rs.3.rs-5035114/v1

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Biallelic mutations in SHMT2 cause neurodevelopmental disorders with cardiomyopathy, spasticity, and brain abnormalities (NEDCASB; OMIM: 619121). This recently described metabolic disorder are characterized by severe intellectual disability, microcephaly, spastic paraplegia, peripheral neuropathy, corpus callosum dysgenesis, facial and limb deformities, and progressive hypertrophic cardiomyopathy. Herein we describe the clinical characteristics of a patient with novel compound heterozygous SHMT2 missense variants (c. 1274G > A: p.R425Q and c.1042C > T: p.R348W), presenting with mild intellectual disability, corpus callosum dysgenesis, and speech delay. Different from previous cases, our patient represents the mildest phenotype reported to date, and expand the phenotypic spectrum of disease associated with SHMT2 mutation.

SHMT2

NEDCASB

Neurodevelopmental disorders

Genetic disease

Folate metabolism is indispensable for highly proliferative physiological processes such as embryonic development¹ and is crucial for maintaining neuronal plasticity and integrity. The SHMT2 gene is located in the 12q13.3 region and encodes serine hydroxymethyltransferase 2, a key catalytic regulator involved in the serine/glycine conversion with a relatively stable structure. It can catalyze the reaction of serine and tetrahydrofolate (THF),transferring a one-carbon unit from serine to tetrahydrofolate, and finally generating 5,10-methylene-THF and glycine^2–4.This is a crucial step in folate metabolism, acting as the first rate-limiting enzyme in the mitochondrial one-carbon metabolism pathway. Dysfunction of SHMT2 can lead to reduced levels of folate metabolism and an altered glycine/serine ratio, resulting in severe neurodevelopmental disorders and cardiac defects. In the mitochondrial one-carbon metabolism pathway, SHMT2 primarily affects the folate metabolism cycle, regulating epigenetic modifications and influencing various amino acids and folate metabolism, multiple mitochondrial functions, protein translation, and nucleotide synthesis^3,5,6.

Loss of function of SHMT2 can lead to neurodevelopmental disorders with cardiomyopathy, spasticity, and brain abnormalities (NEDCASB; OMIM: 619121). However, previous studies have only reported six individuals with SHMT2 mutations from five families (P1-P6), among whom, P1 and P2 are siblings. Almost all patients exhibit severe intellectual disability, microcephaly, overall developmental delay, facial and limb deformities, peripheral neuropathy, and spastic paraparesis. MRI scans demonstrated corpus callosum hypoplasia, and echocardiograms reveales different forms of cardiac defects^7,8(Table 1). Considering the wide range of functions of SHMT2, there may be clinical heterogeneity, and more patient reports are urgently needed.

Table 1

Clinical and molecular findings of individuals with *SHMT2* variants.
Findings Proband ID	Present study	P1	P2	P3	P4	P5	P6
Gender	Male	Male	Male	Male	Female	Female	Male
Variants (NM_005412.6)	c.1274G > A: p.R425Q c.1042C > T: p. R348W	c.1133A > G p. (Asp378Gly)	c.1495C > G p. (Pro499Ala)	c.1495C > G p. (Pro499Ala)	c.1267G > A p. (Gly423Ser)/ c.1124_1125delinsA p(Gly375del)	c.1304A > C p. (Gln435Pro)/ c.469C > T p. (Pro157Ser)	c.1135A > G p. (Asn379Asp)/ c.557C > G p. (Thr186Arg)
Type of variant	Missense	Missense	Missense	Missense	Missense; Frameshift	Missense	Missense
Zygosity	Compound heterozygous	Homozygous	Homozygous	Homozygous	Compound heterozygous	Compound heterozygous	Compound heterozygous
Intellectual disability	Mild	Severe	Severe	Severe	Severe	Severe	NA
Motor delay	-	+	+	+	+	+	+
Cognitive delay	-	+	+	+	NA	+	NA
Speech delay	+	+	+	+	NA	+	+
Seizures	-	-	-	-	-	-	-
Microcephaly	-	+	+	+	+	+	+
Spasticity	-	+	+	+	+	+	+
Thin corpus callosum	+	+	+	+	+	+	+
Cardiac evaluation	-	+	+	+	+	+	+
Facial and limb deformities	-	+	+	+	+	+	+

Herein we identified a male individual with a novel SHMT2 compound heterozygous missense mutation (c.1274G > A and c.1042C > T) through whole exome sequencing (WES). This is the first reported case of an SHMT2 mutation in China. The clinical phenotype primarily manifests as mild intellectual disability and corpus callosum hypoplasia, without microcephaly, or significant motor dysfunction, or cardiac defects. Glycine metabolism levels are within the normal range. Our patient highlights the importance of SHMT2 in neurodevelopment and also demonstrates the diversity of clinical phenotypes in individuals with SHMT2 mutations.

2.1 Plasmid Construction: The cDNA encoding human mitochondrial SHMT2 (NCBI reference sequence NP_005412) was cloned into the pET28a vector, with an additional sumo tag added after the N-terminal His tag. The plasmid was then transformed into the E. coli strain BL21 (DE3). The SHMT2 wt, SHMT2 p.R425Q, and SHMT2 p.R348W plasmids were obtained using the EasyPure Plasmid MiniPrep Kit (TRANSGEN EM101-02).

2.2 Cell Culture and Transfection: HEK293 cells were cultured in DMEM/high glucose medium (Gibco C11995500BT). The cells were maintained in an incubator at 37°C in a humidified atmosphere containing 5% CO2. Cell transfection was performed using Lipofectamine™ 3000 (Thermo). Cell samples were collected at 48 hours.

2.3 SHMT2 Activity Assay: Following the Anti-His beads (Biolinkedin L-1015) method to retain protein activity, the protein was eluted and the samples were ultrafiltered, concentrated, and purified to obtain the enzyme solution. In this assay, DL-β-phenylserine (Sigma 171603) was used as a substitute substrate, and the production of benzaldehyde was subsequently measured at 279 nm, which has strong ultraviolet absorbance. The typical detection mixture in a 1.5 ml centrifuge tube contained 50 mM DL-β-phenylserine, 1 mM EDTA, 25 mM sodium sulfate (Coolaber CS10291), 50 μM PLP (Sigma P9255), 50 mM Hepes buffer (pH 8.0), and enzyme. The rate of benzaldehyde formation was measured using the UV-Vis module of the NanoDrop One (Thermo) at 279 nm and 25°C. The measurement times were 0 min, 5 min, 10 min, and 15 min.⁹

2.4 Genetic analysis: We used whole exome sequencing technology on peripheral blood collected from the proband and his parents. Genomic DNA was prepared from peripheral blood lymphocytes using the phenol-chloroform method. The patient's DNA sample was subjected to exome enrichment using the Nextera Rapid Capture Exome v1.2 kit (Illumina). Subsequently, sequencing was performed using the HiSeq4000 platform (Illumina), generating 150-bp paired-end reads. Data analysis was conducted using the Enliven Variant Annotation and Interpretation System and Berry Genomics' proprietary Verita Trekker Variant Detection System, with reference to the human genome assembly hg19 (GRCh37). This testing mainly targeted single nucleotide variants (SNVs) within 5 bp of the splicing boundary and insertions/deletions (InDels) within 50 bp of the exon region, excluding mutations with a frequency greater than 1% in databases such as 1000 Genomes, ExAC, gnomAD, and the Chinese Genome Database, as well as non-functional mutations sites (such as synonymous mutations, non-coding region mutations, etc.).Following a thorough evaluation that encompassed pathogenicity prediction (using software such as SIFT, PolyPhen2, and CADD), comparison of clinical symptoms, searches in relevant disease database searches, and reference checks, candidate gene mutation sites were pinpointed. The pathogenicity of these sites was assessed and interpreted according to the American College of Medical Genetics and Genomics (ACMG) guidelines and the ClinGen Sequence Variant Interpretation (SVI) expert group's recommendations. Upon identifying the pathogenic mutation, additional validation of the mutations in the proband's family was conducted using Sanger sequencing.

3.1 Clinical Report

We are studying a child with compound heterozygous mutations in SHMT2, whom comes from a Chinese family with unremarkable family history. During pregnancy, an ultrasound detected an enlarged head circumference and intracranial hydrocephalus, the child was born more than half a month prematurely. At birth, the head circumference (HC) was 33 cm (normal range 30-33 cm), the length was 46 cm (normal range: 43-49 cm), and the weight was 2.55 kilograms (normal range: 2.4-2.6 kilograms). Signs of developmental delay were first noticed when the patient began walking at two years old. He was diagnosed with speech delay, and at three years old, this amino acid metabolism levels were normal. MRI scans showed delayed white matter development, corpus callosum hypoplasia, and an enlarged cisterna magna in the same year.

The patient is currently 13 years old and presents whit speech delay, unclear pronunciation, and poor language expression ability, but normal motor abilities. He can participate in sports activities such as basketball and manifesting as learning difficulties but he can play electronic games normally. There were no history of seizures or limb tremors. The head circumference is 55 cm, within the normal range (54-58 cm). No hypotonia was found in the patient, and there were no facial or limb deformities, nor cardiac defects.

3.2 Genetic analysis

We collected peripheral blood from the proband and his unaffected parents, extracted DNA samples, and then performed whole exome sequencing (WES). Sequencing results showed that sequences with a depth of 20X or more accounted for 95.37% in the patient, 95.13% in the father, and 95.44% in the mother. Through data comparison, quality control, and annotation, we identified two SHMT2 compound heterozygous mutations in this patient (missense mutations c.1274G>A: p.R425Q in exon 10 and c.1042C>T: p.R348W in exon 9), which were validated by Sanger sequencing. The results showed that the c.1274G>A mutation was inherited from the father, and the c.1042C>T mutation was inherited from the mother (Fig.1a,1b).

Using the VarCards database (http://www.genemed.tech/varcards/) to predict the SHMT2 mutation sites (c.1274G>A and c.1042C>T), which includes 23 functional prediction software analysis tools such as SIFT, Polyphen2, CADD, LRT, Mutation Taster, and FATHMM. The mutations are predicted to affect protein structure/function with damaging scores of 0.96 and 0.7, respectively. Twenty-two tools predicted the c.1274G>A missense mutation to be pathogenic, and sixteen tools predicted the c.1042C>T missense mutation to be pathogenic. The gnomAD Exome database (https://gnomad.broadinstitute.org/) shows that the both variants are predicted to be highly conserved and deleterious, rare in population databases, and no homozygous occurrences have been reported.

According to the ACMG guidelines and ClinGen SVI expert group's recommendations^10–12, the SHMT2 c.1274G>A and c.1042C>T mutations are classified as variants of uncertain significance(c.1274G>A:PM1+PP3+PM2_Supporting,c.1042C>T:PM1+PM2_Supporting). Comparative analysis of the mutated proteins (c.1274G>A: p.R425Q and c.1042C>T:p.R348W) with wild-type proteins using I-Mutant (http://folding.biofold.org/i-mutant/imutant2.0) demonstrated changes in amino acid size and charge, The wild-type residues are positively charged, whereas the mutant residues are neutral. And the molecular structure diagram of SHMT2, predicted by Alphfold2, reveals that a mutation at R348 results in the loss of all hydrogen bonds, while a mutation at position 425 leads to an increase in hydrogen bonds (Fig.1c), These changes might reduce the stability of the mutated protein and affect SHMT2 enzyme function.

3.3 The SHMT2 R425Q and SHMT2 R348W mutants retained some enzymatic activity

To ascertain if the SHMT2 compound heterozygous mutation in the patient retains any enzymatic activity, we utilized DL-β-phenylserine as an alternative substrate under identical reaction conditions. Given that benzaldehyde exhibits strong ultraviolet absorbance at 279 nm, we transfected HEK293 cells with the SHMT2 R425Q and SHMT2 R348W mutants, extracted the proteins, and conducted a comparative analysis with the WT SHMT2 protein. At intervals of 0, 5, 10, and 15 minutes under consistent reaction conditions, our findings revealed that the R348W mutation diminished SHMT2 activity, and the R425Q mutation also reduced SHMT2 activity (Fig.2). Moreover, as the reaction progressed, the absorbance at 279 nm in the gene mutation reaction system gradually increased until the enzyme was depleted or the substrate DL-β-phenylserine in the reaction system was exhausted. This suggests that although the protease activity of the mutant proteins in degrading DL-β-phenylserine is diminished, a partial activity is still preserved.

We report the first individual in China with a novel SHMT2 compound heterozygous mutation (c.1274G > A: p.R425Q and c.1042C > T: p.R348W) identified through WES. Reportedly, the SHMT2 mutation can affect multiple metabolism pathway in mitochondria, so can leading to a series of severe clinical phenotypes.

To date, including our patient, a total of 9 missense mutations and 1 indel have been reported, all of which are predicted to cause loss-of-function effects^7,8(Fig. 1d).Among them, García-Cazorla et al. reported six patients with SHMT2 gene mutations. These patients mainly presented with intellectual disability and motor dysfunction in the form of spastic paraparesis, peripheral neuropathy, corpus callosum abnormalities, microcephaly, speech delay, facial and limb deformities, and cardiac defects. Majethia et al. reported one case of SHMT2 gene mutation with the main clinical features being developmental milestone delay, intellectual disability, jerky movements of the limbs, multifocal epileptiform abnormalities, microcephaly, facial deformities, and delayed myelination and thinning of the corpus callosum. Compared to the cases mentioned above, our patient also exhibits intellectual disability, corpus callosum abnormalities, and speech delay. However, this patient shows some differences and presents the mildest symptoms among the known cases.

Firstly, while the previously reported patients had severe intellectual disabilities, our patient exhibited only mild intellectual disability. This was manifested as decreased calculation ability and learning difficulties, but he is still able to manage daily life needs and even performs well in some computer games. Secondly, our patient does not exhibit microcephaly. In contrast, among the previously reported six individuals with SHMT2 mutations, all except two, who had normal head circumferences, exhibited microcephaly. Thirdly, it is noteworthy that the previously reported cases all experienced severe spastic paraplegia and hyperreflexia. These patients exhibited a spastic abduction-inner rotation pattern gait, abnormal knee flexion gait, and some even required a wheelchair. However, our patient did not exhibit these phenotypic traits and could even play basketball normally. Most importantly, cardiac defects are a common symptom among previously reported patients with SHMT2 mutations. Five out of six patients exhibited various cardiac defects; however, our patient shows no clear evidence of cardiac involvement.

Based on the enzymatic activity assay of the mutated SHMT2 proteins, the mutated SHMT2 retains partial enzymatic activity, which may compensate for certain cellular functions in the patient and alleviate the severity of the phenotype. This could explain the milder clinical manifestations observed in our patient with the SHMT2 compound heterozygous mutation.

Additionally, all previously reported individuals presented facial and limb deformities, such as arched eyebrows, a tall forehead, shallow orbits, a long philtrum, anteverted nares, low-set thumbs, and two to three toe syndactyly. In contrast our patient did not show these facial and limb deformities; only slow nail growth in the limbs was noted in follow-up records, which required vitamin D3 supplementation. In the previously reported six cases of SHMT2 mutation, blood metabolite tests were within normal ranges, but biochemical tests showed a decreased glycine/serine ratio and reduced folate metabolism. In one of those patients, lactate was found to be high normal^7,8. Our patient, although having normal blood metabolite tests (including amino acid metabolism), might still experience mitochondrial dysfunction due to SHMT2 mutations. The identification of additional families with SHMT2 variants would further help in defining better treatment strategies, prognostication, and informed genetic counseling for the families.

In conclusion, we have reported the first case of SHMT2 mutation in China and characterized the enzymatic activity of the mutated SHMT2. This case presented a clinical phenotype distinct from those previously described, thereby broadening the spectrum of clinical manifestations associated with SHMT2 mutations. Notably, our patient exhibited milder symptoms compared to earlier cases, which can be attributed to the fact that the mutated SHMT2 proteins retain partial enzymatic activity, thereby compensating for certain metabolic pathway functions and leading to a less severe clinical presentation.

Consent for publication

We hereby clarify that written informed consent has been obtained from all participants involved in the study regarding the publication of their personal or clinical details, as well as any identifying images. For minor patients, written informed consent has been obtained from their parents. This consent covers the use of these details and images in this study and in any potential publications in scientific journals.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files. No additional datasets are available.

Ethical approval and consent to participate

This study was conducted in accordance with the Declaration of Helsinki and received approval from the Institutional Review Board (IRB) of Hunan Provincial Maternal and Child Health Hospital. Written informed consent was obtained from the legal guardians of all participants involved. This consent encompasses all aspects of participation and data usage. To protect the privacy of both patients and control subjects, all personal identifiers have been removed from the research data.

Acknowledgments

We extend our gratitude to the child and her family for their support of this study.

Competing Interests

No competing interests have been disclosed by the authors.

Funding

This research was funded by Natural Science Foundation of Hunan Province, China (No.2022JJ40206) and Hunan Provincial Maternal and Child Health Care Hospital Postdoctoral Research Startup Fund.

Authors’ contribution

Conceptualization, Xiao Mao and Hu Pan; methodology, Mei He, Xuan Luo and Juanli Hu; data curation, Hu Pan, Xiao Mao and Zhen Liu; writing—original draft preparation, Hu Pan; writing—review and editing, Zhen Liu, Mei He; supervision, Xiao Mao, Yong Cheng; project administration, Xiao Mao, Zhen Liu; funding acquisition, Xiao Mao and Zhen Liu. All authors have read and agreed to the published version of the manuscript. All authors have read and approved the published version of the manuscript.

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Mild Neurodevelopmental Disorder Due to SHMT2 Mutations: Expanding the Phenotypic Spectrum

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1. Introduction

2. Methods

3. Results

4. Discussion

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