Genetic Analysis and Prenatal Diagnosis of Acute Intermittent Porphyria Caused by Novel Classical Splicing Variant in the Insertion Region of 29-Residue Specific to Human HMBS Protein

doi:10.21203/rs.3.rs-2431452/v1

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Research Article

Genetic Analysis and Prenatal Diagnosis of Acute Intermittent Porphyria Caused by Novel Classical Splicing Variant in the Insertion Region of 29-Residue Specific to Human HMBS Protein

https://doi.org/10.21203/rs.3.rs-2431452/v1

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Background: Acute intermittent porphyria (AIP; OMIM#176000) is caused by mutations in the hydroxymethylbilane synthetase (HMBS) gene, which encodes the third enzyme of the heme biosynthesis pathway. A 29-residue insert (residues 296-324) exists at the interface between domains 1 and 3 of human HMBS (hHMBS), and its function remains unknown. In this study, on the HMBS gene of an AIP pregnant woman from China, we predicted the damage to 29-residue insert regions of a new classical splicing variant, and made prenatal diagnosis of the fetus of this pregnant woman.

Methods: We extracted peripheral blood from pregnant women and their husbands, as well as fetal amniotic fluid cells, and analyzed gene expression using prenatal whole exon sequencing and Sanger sequencing. RNA was extracted from peripheral blood of pregnant women and reverse transcribed into cDNA for alternative splicing verification. The protein structure of abnormal splicing caused by variant was predicted using I-TASSER and PyMOL software. Using AMBER14sb software, we simulated protein molecular dynamics.

Results: Pregnant women and her fetuses have a classical splicing variant in the HMBS gene, c.912+1G>C. The husband of the pregnant woman did not detect the variant. There are no reports of this variant in the literature. Results of peripheral blood transcription tests in pregnant women showed that c.912+1G>C retained the intron 13 and caused the exon 13 skipping. Homology modeling and molecular dynamics analysis revealed that the variant affected the HMBS protein's secondary structure, which led to functional differences.

Conclusion: A new classical splicing mutation with pathogenicity of HMBS gene c.912+1G>C was identified in this study, which expands the molecular heterogeneity of AIP and provides key information for genetic diagnosis.

AIP

HMBS

Splicing Variant

Prenatal diagnosis

Molecular Dynamics

The pathogenic gene HMBS of AIP is located on chromosome 11q23.3. HMBS contains 14 exons, encoding 361 amino acids [1, 2]. AIP is an autosomal dominant genetic disease with low clinical penetrance [3]. There is a predominant involvement of the nervous system and the skin in the clinical manifestations [4]. There are many factors that contribute to the acute attack of AIP, including some drugs, fasting, and hormone changes. Interestingly, all of these factors induce the activity of the first rate-limiting enzyme in the heme biosynthesis pathway, 5'-aminolevulinate synthetase 1 (ALAS1). Many intermediates are produced when liver ALAS1 is induced. In the absence of HMBS enzyme activity, the neurotoxic porphyrin precursors 5‘-aminolevulinic acid (ALA) and porphobilinogen (PBG) accumulate in the liver and are secreted into the plasma and outside the body through the urine [5–9]. AIP incidence rates vary from region to region, and the total incidence rate is about 1.5/100000. It is possible for men and women to get sick, but women are more likely to get sick (accounting for 70% of cases) [10–13]. Most cases of this disease begin between the ages of 20 and 40, with children being much less likely to be affected. At present, the validation of classical splicing variation is currently one of the challenges of genetic diagnosis, especially during prenatal diagnosis. Further genetic diagnosis is possible when conditions permit the evaluation of mutations that alter splicing patterns through functional experiments. Under the condition of obtaining patient cells, transcription experiments represent a feasible option to evaluate mutation's effects on splicing mechanisms. In our study, we aim to evaluate the authenticity and potential consequences of splicing variant c.912 + 1G > C affecting the normal splicing of HMBS. This article discusses the method for determining the pathogenicity of HMBS splicing variant. Additionally, we introduced a new idea for predicting the effect of HMBS variation on protein conformation using molecular dynamics principles.

2.1. Ethical Compliance and Patient Information

The Prenatal Diagnosis Center of the Affiliated Hospital of Inner Mongolia Medical University made a prenatal diagnosis of a pregnant patient. This pregnant woman was previously treated in other hospitals for sudden general paralysis and diagnosed with AIP. According to the pregnant woman, her condition has improved, but she has not undergone genetic testing for AIP related genes. During routine prenatal screening, the fetus did not appear abnormal. The husband of the pregnant woman has a normal phenotype. As part of prenatal diagnosis, we provided genetic counseling to the pregnant woman and her husband, and they signed an informed consent form. A subsequent step was to collect amniotic fluid of pregnant women through amniocentesis, followed by collecting peripheral blood samples from both spouses. The Ethics Committee of Inner Mongolia Medical University approved this study and the patient gave written informed consent to participate in it.

2.2. Prenatal Whole Exome Sequencing and Sanger sequencing

Genomic DNA of amniotic fluid sample was extracted using a trace DNA extraction kit (DK803, Laifeng). The genomic DNA of pregnant woman and her husband extraction was undertaken with the Blood Genomic DNA Extraction Kit (TIANGEN, China). Library preparation and exome capture were performed with Hieff NGS®OnePot DNA Library Prep Kit for Illumina® (YEASEN, China) and xGen Exome Research Panel v1.0 (Integrated DNA Technologies, Inc., United States). Captured libraries were sequenced on HiSeq2500 (Illumina, California, CA, USA).

The quality of the raw data was checked by FASTQC. The clean reads were mapped to the reference genome (GRCh37/hg19) with BWA software. After removing duplicates and base quality recalibration, SNP and Indel variants were called using the GATK pipeline. Detected variants were annotated using ANNOVAR and filtered with minor allele frequencies (MAFs) of < 0.5% for dbSNP, 1000G, ExAC, gnomAD databases. Additionally, copy number variation analysis was performed of probe coverage regions using xhmm and clamms tools.

The interpretation rules of sequence variation data refer to the American College of Medical Genetics and Genomics (ACMG) genetic variation classification standards and guidelines, ClinGen sequence variation interpretation (SVI) and the guidelines for specific genes and diseases.

In order to verify the variant, Peripheral blood DNA from the proband was subjected to Sanger sequencing. The PCR conditions were as follows: a pre-denaturation step (95°C for 5min), followed by 35 cycles of denaturation (95°C for 30s), annealing (60°C for 30s), elongation (72°C for 30s), with a final extension (72°C for 5min). The amplicon was sequenced on the ABI 3730xl Genetic Analyzer (Applied Biosystems, Inc.). Sequences were compared using CodonCode Aligner to reference sequences.

2.3. RT-PCR, PCR and Sequencing

In order to verify the effect of variation on mRNA splicing, total RNA was isolated using the PAXgene Blood RNA Kit (Qiagen#762174). Reverse transcription was performed using Hifair®II 1st Strand cDNA Synthesis Kit (YEASEN Biotech Co., Ltd, China). HMBS-F1: 5’-GTTCCCGCATCTGGAGTTCA-3’ and HMBS-R1: 5’-TGTGCCCCACAAACCAGTTA-3’ were used to amplify the target region of HMBS. The 25ul PCR reaction system included: 0.5ul Taq DNA Polymerase (Thermo Scientific, China), 2.5ul 10× Taq Buffer, 2ul MgCl₂, 0.5ul dNTP, 1ul each primer, 1ul cDNA template and 16.5ul H₂O. PCR cycling conditions were: an initial denaturation of 94°C for 5 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 1 min and then an extra 8 min at 72°C. Successful amplification of the PCR products was confirmed by 2% agarose gel electrophoresis. Then the products were sequenced directly by ABI 3730xl DNA Analyzer.

2.4. The Evolutionary Conservation Analysis of Amino Acid Residues and the Structure Prediction of Mutant Proteins

The protein sequences of different species were downloaded from NCBI. Jalview software was used for multiple sequence alignment and conservation analysis. The I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) was used to simulate the influence of the variable region.

2.5. 3-D Structure Analysis of HMBS

The 3-D structure of the HMBS wild-type (WT) was generated via homology modeling through MODELLER software. The crystal structures of the HMBS housekeeping isoform are available in the Protein Data Bank (PDB) under ID codes 3ECR. The 3-D structure of c.912 + 1G > C mutant were prepared from the amino acid sequences of HMBS wild-type. The resulting structures were structurally optimized by GROMACS version 2021.4 (http://manual.gromacs.org). The AMBER14sb force field was used for all simulations.

2.6. Molecular Dynamics

GROMACS software package was used to simulate the protein molecular dynamics. The protein uses AMBER14sb force field. The protein was loaded into the GROMACS module, and hydrogen atoms and NaCl ions were added. Select TIP3P dominant water model and set periodic boundary conditions. The workflow of molecular dynamics simulation includes four steps: energy minimization, NVT equilibrium, NPT equilibrium and production dynamics simulation. Firstly, the protein heavy atoms were constrained to minimize the energy of water molecules by 10000 steps (including 5000 steps steepest descent method and 5000 steps conjugate gradient method); Then, maintaining the constraints, 50000 step NVT ensemble simulation was carried out for the whole system. The temperature was 298K, and the time step was 2fs; Then 50000 step NPT ensemble simulation was carried out for the whole system, the temperature was 298k, and the time step was 2fs; Finally, the molecular dynamics simulation of the system was carried out in the NPT ensemble for 100ns with a time step of 2fs. The relevant parameters were analyzed by the module of GROMACS software package.

2.7. Molecular docking simulation of HMBS Enzyme Activity

The 3-D structure of the HMBS wild- and mute-type proteins with the dipyrromethane (DPM) cofactor molecule in the center were generated via choosing the 3ECR protein in the PDB as the template. The resulting protein structures were subjected to structural optimization by GROMACS version 2021.4 (http://manual.gromacs.org). The Charmm36 force field was used. The interaction of PBG small molecule with the protein were analyzed through the module of GROMACS software package.

3.1. Identification of Novel HMBS Mutation

Prenatal whole exon sequencing identified a heterozygous splicing variant c.912 + 1G > C in exon13 of the HMBS gene of pregnant women and fetuses. The Sanger sequencing method confirmed that the variation was true and reliable (Fig. 1A). It was not detected by the husband of the pregnant woman, and so the fetus inherited the variant from the mother (Fig. 1B). In ExAC and 1000G databases, the variant ID (rs797045752) was not included, and the dbSNP database recorded the variant ID, but not the population frequency. This variant population is not included in the gnomAD database, and there is no literature report on it.

3.2. Corroboration of HMBS Mutations as the Determinant of Alternative Splicing in Splicing Assay

With primer HMBS-F1/HMBS-R1, the target region of pregnant women and normal controls was amplified by PCR using maternal peripheral blood cDNA as template. Control sample amplification product size is consistent with the theoretical amplification length based on the amplification product of the control sample. As compared to the control sample, the amplification product from pregnant women has two more bands (Fig. 2A). Pregnant women's cDNA amplification products and control samples are sequenced using Sanger sequencing methods. It can be seen in band 1 that there is an overlap peak, demonstrating that intron 13 has been retained (by 88 base pairs) in pregnant women's cDNA. The sequencing result for band 2 shows a wild-type sequence. During the band 3 analysis, it was found that pregnant women samples had missing 87 bp in exon 13 as a result of exon 13 skipping (87 bp are missing). (Fig. 2B). As evidenced by the sequencing results of the control sample, exon skipping and intron retention were not observed, indicating that c.912 + 1G > C actually affected HMBS gene splicing (Fig. 2C).

3.3. Model Analysis of Abnormal Structure of HMBS Caused by Mutation

According to the evolutionary conservation analysis of the HMBS amino acid residues, the damaged amino acids are highly evolutionary conserved among species (Fig. 3A, B). The p.H305Vfs*31 causes HMBS protein to shift from the 305th amino acid and terminates translation in advance. HMBS: p.H305Vfs*31 protein undergoes a greater than 40% sequence change in its Domain3 region and the length of α helix in this region is shortened, thereby decreasing protein stability. The p.276_304del leads to amino acid deletions from 276 to 304 of HMBS protein. HMBS: p.276_304del protein causes more than 20% of the Domain3 region to be deleted, and the length of α helix in this region is reduced, which may reduce protein stability (Fig. 3C). In the absence of molecular dynamics analysis, PyMol and modeler homologous modeling can only simulate the conformational changes of p.276_304del and p.H305Vfs*31 in domain 3 in comparison to WT. Domains 1 and 2 of p.276_304del and p.H305Vfs*31 are not significantly different from those of WT. Based on this result, homologous modeling without molecular dynamics analysis can only simulate local changes caused by mutation, but not the overall conformational impact of mutation.

3.4. Protein molecular dynamics simulation of HMBS

According to the analysis results, the root mean square deviation (RMSD) of p.276_304del fluctuates the most, indicating this mutation has the greatest effect on the protein's overall conformation. Conformational changes in WT and p.H305Vfs*31 are similar. Compared to p.276_304del and p.H305Vfs*31, WT has greater stability, suggesting that p.276_304del and p.H305Vfs*31 influence protein conformation more or less (Fig. 4A).

WT, p.276_304del, and p.H305Vfs*31 have the same first 270 amino acid residues, so it is possible to compare their root mean square fluctuation (RMSF). From the analysis results, it is evident that p.H305Vfs*31 fluctuates most in this region, especially between 180–200 and 250–270. There is a similar difference between residue fluctuations in p.276_304del and p.H305Vfs*31 for other comparable regions. As a result of this, we can infer that p.276_304del and p.H305Vfs*31 significantly affect the conformation of residues in 180–200 and 250–270 (Fig. 4B).

Radius of gyration (Rog) measures protein conformation compactness. As can be seen from the results of the analysis, the Rog of WT is the lowest, indicating the compactest structure. Among the three, p.276_304del has the largest Rog and fluctuates the most, indicating looser structure than WT and p.H305Vfs*31. Rog's results are consistent with those of RMSD, indicating that p.276_304del makes the protein conformation more loose, and further may affect its function (Fig. 4C).

Solvent accessible surface area (SASA) stands for surface area of protein in contact with solvent. In general, the higher the SASA value, the greater the contact area between the protein and the solvent, which results in more internal structure exposure. The SASA analysis results show that after 80ns of molecular dynamics simulation, the SASA values of WT and p.276_304del are equal and low. As p.H305Vfs*31 has a relatively high SASA value, its internal domain is exposed more, which may affect its function (Fig. 4D).

It is clear from the analysis results of hydrogen bond differences that WT is compact and undamaged, and its protein has significantly more hydrogen bonds than p.276_304del and p.H305Vfs*31. As a result of the reduction in internal hydrogen bonds of p.276_304del and p.H305Vfs*31, structural integrity and compactness are negatively affected, which could further contribute to functional problems (Fig. 4E).

According to secondary structure analysis, the most significant change caused by p.276_304del and p.H305Vfs*31 is the composition of protein secondary structures. Compared to p.276_304del and p.H305Vfs*31, WT has a significantly higher secondary structure content. As a result of having only 200 amino acids in its secondary structure, p.276_304del has the least secondary structure. As far as the number of elements in the secondary structure goes, α helix has the largest number. As compared with p.276_304del and p.H305Vfs*31, WT has 110 acting residues that can form α helix, whereas p.276_304del and p.H305Vfs*31 only have 100 α helix. As well as these differences of α helix, there are differences in the composition of other secondary structures between WT, p.276_304del, and p.H305Vfs*31. It is likely that the variation of p.276_304del and p.H305Vfs*31 leads to a change in the secondary structure of the protein, which may lead to functional differences (Fig. 4F).

3.5. Modeling mutations induced abnormalities in enzyme activity of the HMBS enzyme through molecular interactions

According to the homologous modeling results obtained after molecular dynamics analysis, p.276_304del and p.H305Vfs*31 have changed significantly compared to WT in many regions. The molecular dynamics technique allows the conformational motion of simulated proteins to change over time to reflect more atomic details. After molecular dynamics analysis, homologous modeling is performed, and the conformational change of simulated mutant proteins is closer to the real situation (Fig. 5A, B, C).

The structure of HMBS proteins is altered by mutations. In order to determine whether mutant proteins p.276_304del and p.H305Vfs*31 are enzymatically active, we used computer simulations of their interactions with PBG small molecules. As a result of the results, WT strongly interacted with PBG small molecules, and the binding site was located close to the active center of DPM (Fig. 5A). However, PBG small molecules are not only far from the DPM active centers of p.276_304del and p.H305Vfs*31, but even almost unable to interact with both (Fig. 5B, C). Based on our findings, it is likely that these two mutant proteins are almost completely inactive.

Our study found a new mutation affecting splicing in the HMBS gene in a pregnant woman with AIP and her fetus, which was located at the junction of exon 13 and intron 13. In most human genes, the coding regions are discontinuous and contain multiple exons. mRNA precursors are produced after transcription of the gene. Splicing of mRNA precursor transcripts occurs when introns are cut from the transcripts, and then exons are spliced together to create functional gene molecules. In order to initiate the splicing reaction, the splice body must identify the splice site at the exon-intron junction, including the splice donor located at intron 5’ and the splice receptor located at intron 3’ [14, 15]. Almost all introns contain GT and AG boundary sequences, which are crucial for splice recognition [16, 17]. As shown in our study, c.912 + 1G > C caused damage to the 5' splice donor in intron 13 by damaging its 5' splice. As a result of the misrecognition of the GT position of exon 13, 87 nucleotides were cleaved at the end of exon 13. Because the splice misread the GT position of exon 13, 87 nucleotides were cleaved leaving a HMBS protein lacking 276–304 amino acids. A second finding in the study was that c.912 + 1G > C interferes with splice recognition, resulting in production of a HMBS protein that retains intron 13. The HMBS truncated protein contains 304 normal amino acids and 30 abnormal amino acids because of a premature termination codon tag inserted at nucleotide 88 downstream of exon 13 of the HMBS gene. In principle, introducing a mutation to the stop codon early in protein synthesis will result in a truncated protein, thereby affecting protein folding and stability. In Swedish patients, the most common HMBS gene mutation (Trp198Term) results in proteins that are severely truncated, possibly inactive, and easily degradable. AIP has a low penetrance (8%), so patients with this mutation have the highest incidence rate. In our study, p.H305Vfs*31 caused the HMBS protein to shift from the 305th amino acid, and halted translation early. It is estimated that the sequence change in domain 3 of p.H305Vfs*31 exceeds 40%, which will interfere with the folding of the enzyme during biosynthesis and destroy its stability. We can, however, observe normal splicing of HMBS gene mRNA in the peripheral blood of pregnant women, and the normal subtype bands are much stronger than the abnormal subtype bands. As a result, we suggest wild-type HMBS protein possesses a dominant role in pregnant women, which may be a direct indication that the symptoms aren't very serious.

There are three domains in the HMBS protein. The residues 1-116 and 216–239 of domain 1 constitute a discontinuous domain. Domain 2 consists of residues 117–215, while domain 3 consists of residues 240–361 [18]. By way of the flexure hinge region, the three domains are connected. The active site of DPM cofactor is located at the interface between domains 1 and 2 [19]. By combining HMBS with DPM cofactor, in the same catalytic position, four repeated reactions are performed to catalyze the formation of porphyrin. The domain 3 consists of an antiparallel β-sheet with three chains, one of which is covered by three α-helixes. Domain 3 interacts equally with domains 1 and 2, contributing to overall protein stability [20]. In contrast to Escherichia coli (EcHMBS) and Arabidopsis thaliana (AtHMBS), domain 3 of hHMBS contains 29-residue (residues 296–324) on the interface between domain 1 and 3. The 29-residue inserts are far away from the active site, and their functions are still unknown. Since these 29-residue inserts are sandwiched between domain 1 and domain 3, they may push domain 3 towards domain 2 and prevent them from interacting massively. Accordingly, we speculate that hHMBS has a lesser movement of the domain compared to EcHMBS because of the 29-residue present [19]. Moreover, we speculate that the interaction between 29-residue and domain 1 may regulate conformational fluctuations related to enzyme interactions, resulting in weak dimerization of the crystal. As seen in our study, defects in domain 3 of p.276_304del and p.H305Vfs*31 adversely affect the integrity of the 29-residue region of hHMBS, thus affecting the overall stability of hHMBS. Overall, we speculate that the defects in residues 296–324 of p.276_304del and p.H305Vfs*31 may indirectly affect hHMBS catalyzed porphyrinogen formation rate.

Catalyzed by HMBS using the same catalytic site four times, the extension of polypyrrole can be achieved. It is important that the intermediate of the reaction be transferred from the active site on time, which is closely related to the flexible sliding ring E250-C261 [21]. We emphasize the importance of the flexible sliding ring, which is critical to reaction extension. There are seven residues (E250, A252, L254, H256, E258, G260 and C261) in the slip ring. When these residues are mutated, they could lead to AIP, which could limit the fluidity of the ring, limiting reaction efficiency. As a result of our present study, it is evident from RMSF analysis that residue fluctuations in p.276_304del and p.H305Vfs*31 within the 250–270 region are significantly greater than in WT. There is a possibility that this could be due to damage to the 29-residue insert in p.276_304del and p.H305Vfs*31. If the 29-residue insert is impaired, its restrictions on the movement of domain 3 are reduced, resulting in an increased degree of freedom for flexible sliding ring, which may reduce the reaction's efficiency. Furthermore, the RMSF analysis of the active site loops (residues 56–76) of WT, p.276_304del and p.H305Vfs*31 showed more significant differences between them. The RMSF values of p.276_304del and p.H305Vfs*31 were significantly lower than those of WT, and the difference between p.H305Vfs*31 and WT was the greatest. In light of this, we speculate that our analysis may be corroborated in further detail. When 29-residue insert is damaged, flexible sliding ring is left more free to react during polypyrrole extension, reducing the efficiency of the response of the active site loops to respond, which in turn reduces enzyme activity indirectly.

In addition to forming the main chain connection between domain 1 and domain 2, 29-residue in domain 3 is also involved in filling domain 1, and this interaction is mainly mediated by hydrogen bonds [20]. The residues 312–315 and 318–321 form antiparallel chains. When the chain is bifurcated, the hydrogen bonds of the main chain Leu315NH-Ile318CO and Leu315CO-Ile318NH are interrupted and the main chain-side chain interaction Asp312CO-Arg321NH takes their place [20]. The carbonyl oxygen of Val316 forms a hydrogen bond with the side chain of Arg251. Leu315, Val316, Ile318 and Ala320, the hydrophobic side chains of 29-residues, interact with Leu329, Ile248 and Leu244 of domain 3. Furthermore, hydrogen bonds between Gly317CO-Ile110NH and Thr319NH-Ile110CO, hydrogen bonds between the side chain of Gln314 and the carbonyl group of Phe108, and hydrogen bonds between OG atoms of Thr109 and Thr319 stabilize the interaction between domains 1 and 3 [20]. Our study finds domain 3 of p.H305Vfs*31 to be completely abnormal from the 305th residue, indicating that the interaction between domains 1 and 3 of p.H305Vfs*31 is much weaker than what is found in wild-type.

There are hydrophobic interactions among some residues in the three domains of HMBS [20]. As an example, residues Leu97, Leu244 and Cys247 are between domains 1 and 3, residues Ala152, Met212 and Leu285 are between domains 2 and 3, and residues Trp283 and Gln153 are between domains 2 and 3 [20]. In our study, p.276_304del lost the key sites Leu285 and Trp283 involved in maintaining domain 3 interactions. Because of this, compared with WT, p.276_304del has reduced hydrophobicity, which makes it harder for the protein to fold inward to form secondary structure, and further to form domains and tertiary structures. In addition, the decrease of hydrophobicity does not contribute to the formation of α-helix, which affects the protein's stability. Additionally, Leu245 in domain 3 is a conservative hydrophobic side chain that interacts with Pro241, Pro302 and Pro327 in domain 1. The replacement of Leu245 by Arg245 related to AIP will bury a large positively charged side chain, affecting the stability of domain 3 folding [20]. Our study shows that p.276_304del lacks Pro302, p.H305Vfs*31 lacks Pro327, and both p.276_304del and p.H305Vfs*31 lack the key interaction sites that interact with Leu245, which will adversely affect the folding stability of domain 3.

In the 29-residue insert, far from the active center, the c.912 + 1G > C mutation produces p.276_304del and p.H305Vfs*31 with defective amino acid regions. According to our computer simulations, the PBG small molecule was unable to bind to the DPM active centers of p.276_304del and p.H305Vfs*31 and the catalytic reaction could not proceed. As a result, we predict that p.276_304del and p.H305Vfs*31 will lose their catalytic activity completely. There was previously no clear understanding of the role of the 29-residue insert region of the hHMBS protein. We found that the 29-residue insert plays a crucial role in keeping the conformational stability of the hHMBS protein, so that its failure could result in a complete loss of enzyme activity.

AIP can primarily be diagnosed clinically by the elevation of urinary ALA and PBG, but these levels are not always elevated in the asymptomatic population. Therefore, the importance of genetic testing to diagnose AIP in asymptomatic patients, especially in prenatal diagnosis, cannot be overstated. There is a high level of genetic diversity and familial transmission of the HMBS gene mutation. There is a good chance that other members of the family will have the same mutation site when a family screening is conducted for AIP patients. In order to reduce the acute attack of AIP, these potential AIP patients or their guardians should be advised to avoid various inducements of AIP. In the current literature, AIP cases are mostly reported on their clinical manifestations and treatments, but our study completed prenatal diagnosis of AIP families for the first time. In accordance with ACMG guidelines, we rated the HMBS: c.912 + 1G > C as "pathogenic." The evidence supporting the pathogenicity rating is as follows. PVS1_Strong: functional deletion is one of the pathogenic mechanisms of HMBS gene abnormality, which is a classical splice site variation that may cause functional loss of the gene. PS3_Moderate: in functional experiments, this mutation affects splicing, resulting in HMBS intron 13 retention and exon 13 skipping. PM2_Supporting: the variation is a rare one, and the frequency of the East Asian general population is not included in the gnomAD database. PM4: insertion of a non-repetitive region alters the length of the protein. PP3: bioinformatics software predicts that splicing is affected by variation. PP4: Family history or phenotype of the mutation carrier are highly consistent with AIP. After the follow-up visit, we learned that the pregnant woman had given birth to a baby girl at 36 weeks of pregnancy. The baby girl is in good health at present, but she did have a brief episode of apnea after birth, as we know.

In conclusion, we found and verified a new HMBS splicing variant c.912 + 1G > C (p.276_304del and p.H305Vfs*31). Meanwhile, as NM_000190: c.912 + 1G > C variant is currently cited as "pathogenic" change in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/RCV002293282.1/) databases, our study strongly supports experimentally a pathogenic effect.

AIP

Acute intermittent porphyria

HMBS

Hydroxymethylbilane synthetase

ALAS1

5'-aminolevulinate synthetase 1

ALA

5‘-aminolevulinic acid

PBG

porphobilinogen

GRCh37

Genome Reference Consortium Human Build 37

dbSNP

Database of single nucleotide polymorphisms

1000G

1000 Genome Project

ExAC

Exome Aggregation Consortium

gnomAD

Genome Aggregation Database

Wild-type

ACMG

American College of Medical Genetics and Genomics

ClinGen

Clinical Genome Resource

PDB

Protein Data Bank

NVT

Canonical ensemble

NPT

Constant-pressure, constant-temperature

DPM

Dipyrromethene

RMSD

Root mean square deviation

RMSF

Root mean square fluctuation

Rog

Radius of gyration

SASA

Solvent accessible surface area.

Ethics Approval and Consent to Participate: The study protocol was approved by the ethics committee of Inner Mongolia Medical University. The study consent forms were attained from the participants and all participants signed informed consent forms.

Consent for Publication: Consent to publish has been obtained from participants to report individual patient data.

Availability of data and materials: The datasets for this study can be found in the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/RCV002293282.1/).

Competing Interests: The authors declare that they have no competing interests.

Funding: This study was supported by the National Natural Science Foundation of China (No. 81960130), Scientific and technological Innovation team of Inner Mongolia Medical University, China (No. YKD2022D010) and Key research projects of Inner Mongolia Medical University, China (No. YKD2021ZD008).

Authors’ contributions: Conceptualization and designed the experiments: L.L., formal analysis and investigation: H.W., writing—original draft preparation: L.L., writing—review and editing, L.Z. and J.Z., resources: H.M., supervision, J.Z., funding acquisition: L.L. and J.Z. All authors have read and agreed to the published version of the manuscript.

Acknowledgments: The authors extend deep felt gratitude to the patients and their willingness to participate in this study.

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Genetic Analysis and Prenatal Diagnosis of Acute Intermittent Porphyria Caused by Novel Classical Splicing Variant in the Insertion Region of 29-Residue Specific to Human HMBS Protein

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Ethical Compliance and Patient Information

2.2. Prenatal Whole Exome Sequencing and Sanger sequencing

2.3. RT-PCR, PCR and Sequencing

2.4. The Evolutionary Conservation Analysis of Amino Acid Residues and the Structure Prediction of Mutant Proteins

2.5. 3-D Structure Analysis of HMBS

2.6. Molecular Dynamics

2.7. Molecular docking simulation of HMBS Enzyme Activity

3. Results

3.1. Identification of Novel HMBS Mutation

3.2. Corroboration of HMBS Mutations as the Determinant of Alternative Splicing in Splicing Assay

3.3. Model Analysis of Abnormal Structure of HMBS Caused by Mutation

3.4. Protein molecular dynamics simulation of HMBS

3.5. Modeling mutations induced abnormalities in enzyme activity of the HMBS enzyme through molecular interactions

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

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