Unraveling the Genetic Mysteries of Spinal Muscular Atrophy in Chinese Families

doi:10.21203/rs.3.rs-4770645/v1

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Research Article

Unraveling the Genetic Mysteries of Spinal Muscular Atrophy in Chinese Families

https://doi.org/10.21203/rs.3.rs-4770645/v1

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Objective

Spinal muscular atrophy (SMA) is a motor neuron disorder encompassing both 5q and non-5q forms, causing muscle weakness and atrophy due to spinal cord cell degeneration. Understanding its genetic basis is crucial for genetic counseling and personalized treatment options.

Methods

This study analyzed families of patients with suspected SMA at our institution from February 2006 to March 2024. Various molecular techniques, including multiplex ligation-dependent probe amplification (MLPA) analysis, long-range polymerase chain reaction (PCR) combined with nested PCR, Sanger sequencing, and whole-exome sequencing (WES), were employed to establish a thorough genetic variant profile in 680 Chinese pedigrees with clinically suspected SMA.

Results

Out of 680 families suspected of having SMA, 675 exhibited the SMN1 gene, while three families were linked to the IGHMBP2 gene. One family exhibited a genetic variation in the NEB gene, and another family exhibited a variation in the SCO2 gene. Among the families with mutations in the SMN1 gene, 645 families exhibited either E7‒E8 or E7 homozygous deletion. Some families displayed E7‒8 heterozygous deletion along with other mutations, such as E1 or E1‒6 heterozygote deletion and point mutations. Furthermore, one family demonstrated a compound-heterozygous double mutation, while another family carried a type "2 + 0" mutation alongside a point mutation.

Conclusions

This study comprehensively analyzed the genetics of suspected familial SMA cases in the Chinese population, providing insights into the molecular genetic mechanisms of SMA and the utility of various detection techniques. The findings reveal important implications for genetic counseling, prenatal diagnosis, and targeted therapies in clinical practice.

Spinal muscular atrophy

5q SMA

non-5q SMA

SMN1 gene

Genetic diagnosis

Prenatal diagnosis

Spinal muscular atrophy (SMA) is a hereditary disorder characterized by the degeneration of anterior horn cells, resulting in muscle weakness and atrophy ^1,2. Various types of SMA, such as 5q and non-5q SMA, have been linked to a range of genetic mutations.

The most severe clinical and genetic form of SMA, known as 5q SMA, is linked to mutations in the SMN1 gene located on chromosome 5q13.2. This autosomal recessive genetic disorder encompasses various subtypes, including type 0, type 1 (Werdnig-Hoffmann disease), type 2 (Dubowitz syndrome), type 3 (Kugelberg-Welander disease), and type 4 (adult-onset SMA) ^3–6. The global prevalence of this ailment is estimated to be 1 in 10,000 individuals, with a carrier frequency of 1 in 50 in the general population ^3,7. 5q SMA was historically the most common genetic cause of infant mortality; however, this changed in 2016 with the advent of three novel treatments: Nusinersen, Zolgensma, and Risdiplam ³.

Non-5q SMA is characterized by its infrequency, genetic heterogeneity, clinical variability, and lower prevalence compared to 5q SMA ⁸. This condition is linked with pathogenic mutations in genes such as IGHMBP2, PLEKHG5, GDAP1, ASAH1, ATP7A, TRPV4, VCP, and others ^2,8–10. Progress in diagnostic methodologies has resulted in an increasing number of clinically diagnosed cases. Presently, there is no established treatment for non-5q SMA.

The advancement of technology has led to an increasing array of genetic testing methods for the detection of SMA. These methods differ in complexity, cost, reliability, and speed. It has been observed that approximately 95% of individuals with 5q SMA exhibit a deletion of exon 7 of the SMN1 gene or the c.840C > T variation between SMN1 and SMN2 ^1,11. The literature discusses various detection methods, including fluorescent quantitative PCR (FQ-PCR), multiplex ligation-dependent probe amplification (MLPA), PCR-restriction fragment length polymorphism (PCR-RFLP), PCR denaturing high-performance liquid chromatography (PCR-DHPLC), and digital PCR (dPCR), among others ^11–17. The integration of long-range PCR and nested PCR, or third-generation sequencing, has been employed to identify the residual 5% of 5q SMA cases caused by point mutations ^17,18. Furthermore, next-generation sequencing methods, including whole-exome sequencing (WES), have displayed efficacy in detecting non-5q SMA cases ^8,10.

The genetic background of SMA can be complex, and the absence of a large family cohort hampers a comprehensive understanding of condition ¹. In this research, to the best of our knowledge, we presented the largest cohort of SMA in China and shared the insights acquired through the diagnostic process. This result significantly contributes to the global SMA genetic dataset and enhances testing protocols. This study addressed the question of what subsequent genetic analysis clinicians should perform upon suspecting SMA in a patient.

Study cohort

Between February 2006 and March 2024, a total of 680 families with suspected SMA patients were recruited from the Genetics and Prenatal Diagnostic Center of the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). This study received approval from the Ethics Committee of the First Affiliated Hospital of Zhengzhou University, and all participants provided voluntary informed consent before their inclusion.

Pregnant women underwent villus sampling at 12‒14 weeks or amniotic fluid retrieval at 16‒20 weeks for a prenatal diagnosis. The decision to undergo prenatal diagnosis was entirely voluntary for any family.

DNA extraction

DNA was extracted from human peripheral blood, amniotic fluid, and chorionic villi using the Qiagen Genomic DNA Extraction Kit or a tissue extraction protocol on an Eppendorf epMotion 5075 m workstation. The DNA concentration was determined using the Life Technologies Qubit dsDNA HS Assay Kit.

MLPA

The number of SMN1 and SMN2 gene copies was determined using the SALSA P060-B2 SMA Kit from MRC Holland. PCR products were analyzed on an ABI 3130 Genetic Analyzer and processed with Coffalyzer software. Ratios falling below 0.7, between 0.7 and 1.3, 1.3 and 1.7, and between 1.7 and 2.3 indicated one, two, three, and four gene copies, respectively.

Long-range PCR, nested PCR, and Sanger sequencing

Long-range PCR was performed using the KOD FX kit to amplify a 28.2-kb region containing exons 1‒8 of SMN1. Subsequently, nested PCR was used to amplify each SMN1 exon using 1 µL of the initial product as a template. The resulting product was purified and sequenced using the ABI 3130 Genetic Analyzer ¹⁸.

WES

Genome DNA quantification was performed using Qubit 4.0. DNA libraries were constructed using the Ada and Index Kit and the Enzyme Plus Library Prep Kit. Exon coding and splicing regions were captured with the AIExome Human Exome Panel V2 Plus, followed by sequencing on the NovaSeq6000 platform. Paired-end reads were aligned to the human reference genome GRCh37/hg19 using Sentieon software following GATK guidelines. Duplicate reads were removed with Picard version 2.9.0, and variants were annotated using multiple databases. Candidate gene variants were validated through Sanger sequencing.

An overview of the enrolled families

A total of 680 Chinese families with suspected SMA patients were recruited for the study. The genes involved and the types of mutations are presented in Table 1. A total of 675 families exhibited an association with the SMN1 gene, whereas the IGHMBP2 gene was linked to three families, and the SCO2 and NEB genes were each identified in one family. The mutations identified in the IGHMBP2 gene were E628* and A786Pfs*45, K220R and R637L, and R443C and A600V, respectively. The mutations identified in SCO2 were F237fs and A97_Q101dup, while those identified in NEB were R8358Vfs*20 and E8167*.

Table 1

Summary of genetic variation data for 680 families. E7‒8 indicates that exons 7 and 8.
Gene	NM	Variation type	Number of families
SMN1	NM_000344	Homozygote deletion in E7‒8 or E7	645
		E7‒8 heterozygote deletion combined with E1 heterozygote deletion	1
		E7‒8 heterozygote deletion combined with E1‒6 heterozygote deletion	1
		Heterozygote deletion in E7‒8 accompanied by single-point mutation	26
		"2 + 0" genotype accompanied by single point mutation	1
		Compound-heterozygote mutations	1
IGHMBP2	NM_002180	Compound-heterozygote mutations	3
SCO2	NM_005138	Compound-heterozygote mutations	1
NEB	NM_001164508	Compound-heterozygote mutations	1

Families related to the SMN1 gene

Our research revealed that a significant proportion of families (94.8%, 645/680) exhibited homozygous deletion in SMN1 E7‒8 or E7, as depicted in Table 1 and Fig. 1. Only two families exhibited E7‒8 heterozygous deletion combined with E1 or E1‒6 heterozygote deletion. Among the remaining families (4.1%, 28/680), point mutations in SMN1 were observed. Notably, one family displayed a compound-heterozygous double mutation (Table 1).

In a cohort comprising 645 Chinese families with a homozygous deletion in SMN1 E7‒8 or E7, the predominant patient genotypes were identified as 0-0-3-3 (46.0%), 0-0-2-2 (33.9%), and 0-0-4-4 (4.7%), as illustrated in Fig. 1A. The most common carrier genotypes, as depicted in Fig. 1B, were 1-1-2-2 (52.9%), 1-1-3-3 (19.6%), and 1-1-1-1 (14.5%). The genotypes of 2-2-2-2 and 2-2-1-1 may indicate carriers of the "2 + 0" allele.

Among the 28 families with point mutations in SMN1, 14 distinct mutations were identified, as illustrated in Fig. 2. The inheritance of point mutations from parents was identified in affected patients and fetuses without any de novo mutations observed. Twenty-six families exhibited heterozygous deletion in exon 7, along with a single-point mutation. One family demonstrated a compound-heterozygous double mutation comprising the variants N178Tfs*39 and R288M. Another family was identified as carriers of type "2 + 0" and the S8Kfs*23 mutation (Figure S1). The mother possesses a genotype of 2-2-1-1 with an S8Kfs*23 point mutation, while the father potentially possesses a "2 + 0" genotype of 2-2-1-1. The patient exhibits a genotype of 1-1-2-2 with an S8Kfs*23 point mutation, and the brother has a genotype of 3-3-1-1 with the same S8Kfs*23 point mutation.

Asymptomatic persons with homozygous deletion of SMN1 E7

Five asymptomatic individuals were identified in three separate families, denoted by green numbers (Fig. 3), all of whom exhibited homozygous deletion of exon 7 of the SMN1 gene. In Family A, a 40-year-old pregnant woman (II 2) underwent genetic disease carrier screening due to consanguinity between her parents. The screening revealed a homozygous deletion of the SMN1 gene E7. Subsequent MLPA analysis of the family members indicated that both the older sister (42 years old; II 1) and the younger sister (32 years old; II 3) shared the same genotype as the pregnant woman (II 2). The mother (I 2) and brother (II 4) were carriers of SMA, while the father (I 1) was likely a carrier of SMA "2 + 0" (Fig. 3A). In Family B, the father (I 1) was identified as a carrier, while the mother (I 2), who was 50 years old in 2024, displayed no symptoms. The firstborn son (II 1) succumbed to SMA at the age of five, while the younger son (II 2), who is presently 16 years old, is confined to a wheelchair (Fig. 3B). In Family C, the father (I 1) was identified as a carrier, while the mother (I 2), aged 30 in 2024, exhibited no symptoms. The eldest son (II 1), aged six years, who cannot ambulate, received two injections of Nusinersen. The second fetus was terminated due to SMA (Fig. 3C).

Fascinating discoveries during the detection

Two distinct families were identified as having false positives for MLPA (Fig. 4). In Family A, the child exhibited dysphagia, a choking cough, and muscle weakness since birth. The MLPA analysis confirmed the diagnosis of SMA in the child, attributing it to a homozygous deletion of the SMN1 gene in E7. The mother was identified as a “1 + 0” carrier, while the father was presumed to be a carrier of the "2 + 0" type. However, the sequencing results superseded the MLPA results (Fig. 4A). A single copy of the SMN1 gene was detected in the child, featuring a benign variant c.835-17C > G, leading to a diagnosis of carrier status for SMA. The mother was identified as a “1 + 1” gene type. Subsequent WES results revealed R8358Vfs*20 and E8167* mutations in the NEB gene in the child. Both variants were found to have been inherited from the maternal and paternal lineages, respectively. The fetus in Family A also carried the same two disease-causing mutation sites of the NEB gene. In Family B, the father's MLPA results were inconsistent with the sequencing results. The MLPA analysis indicated the presence of a single copy of SMN1 E7, while the sequencing revealed the presence of two copies (Fig. 4B). The c.835-5T > G of the SMN1 gene is pathogenic.

Two children diagnosed with SMA underwent both MLPA and WES in two separate families (Fig. 5). The MLPA results displayed a pattern of 0-0-3-3 for both individuals. The sequencing depth of the SMN1 gene exon 7 in WES analysis was observed to be below the mean, highlighted by a black arrow, while the SMN2 gene exon 7 exhibited a higher sequencing depth, as denoted by a red arrow.

This study provided a comprehensive analysis of the genetic profile of SMA and presented the most extensive dataset currently known to us. It also discussed the challenges faced during testing and proposes follow-up testing protocols for individuals suspected of SMA and their family members.

In addition to the presence of the SMN gene in most families, other genes were identified. Mutations within the immunoglobulin mu DNA binding protein (IGHMBP2), an RNA-DNA helicase, lead to SMA with respiratory distress type I (SMARD1) and Charcot Marie Tooth type 2S (CMT2S) ¹⁹. Furthermore, mutations in the SCO2 gene result in cytochrome c oxidase deficiency, causing symptoms such as Leigh syndrome, hypertrophic cardiomyopathy, lactic acidosis, ventilator insufficiency, and a phenotype similar to SMA ²⁰. Linear myopathy associated with the NEB gene is a rare congenital neuromuscular disorder characterized by muscle weakness, hypotonia, delayed acquisition of gross motor skills, and diminished respiratory capacity ²¹.

The genotypic diversity observed among 5q-SMA patients highlights the presence of structural variability, including partial gene deletions, point mutations, duplications, or conversions within the SMN1 and SMN2 genes. This report represents a preliminary investigation into Exon 1‒6 deletion (Table 1), identified using the MLPA P021 probe. The genotype 0-0-3-3 exhibits the highest prevalence among patients, while the genotype 1-1-2-2 is most frequently observed among carriers (Fig. 1). It is widely acknowledged that carriers of the genotype 1-1-2-2 give birth to individuals with the genotype 0-0-2-2, as opposed to 0-0-3-3. According to the findings, the majority (73.83%) of the 149 Chinese patients with SMN1 deletion possessed two copies of SMN2 ²². However, the prevalence of the 0-0-2-2 genotype was not found to be the highest in this study. This may be attributed to the death of the SMA-afflicted children (0-0-2-2 genotype) in most families during prenatal diagnosis visits, which were not included in the statistics, resulting in the underrepresentation of this genotype.

Genotypes like 0-1-3-2, 0-2-3-1, 1-2-3-2, 1-2-1, and others may indicate the presence of single-nucleotide polymorphisms of SMN2 and SMN2/SMN1 hybrid genes ²³. In families with homozygous deletion of exon 7, most parents are carriers with only one copy of SMN1, while a minority exhibit the presence of two copies of SMN1 (Fig. 1). Our previous research has demonstrated that among fathers possessing two copies, some exhibit a normal “1 + 1” genotype, while others display a "2 + 0" genotype ¹². Kirwin et al. reported a homozygous double mutation in an SMA patient whose parents were consanguineous, specifically identified as p.Val19fs*24 and p.Pro221Leu ²⁴. In this research, we documented the presence of a heterozygous double mutation of N178Tfs*39 and R228M in an SMA patient whose parents were not blood-related (Fig. 2). Moreover, the N178Tfs*39 variant is inherited from the father, while the R228M variant is inherited from the mother.

The variability in the copies and structural composition of the SMN2 gene among individuals may contribute to the heterogeneity observed in the clinical presentation and severity of 5q-SMA patients ²⁵. Previous studies indicate that the number of SMN2 copies is inversely related to the severity of the disease, and the presence of the SMN1/SMN2 hybrid gene is associated with patients exhibiting a relatively mild course of disease ^26,27. Discrepancies in the number of E7 and E8 segments within the SMN1 or SMN2 gene, as revealed by MLPA analysis, may suggest the formation of hybrid genes. Sanger sequencing analysis identified a mutation in the SMN2 gene (NM_01741.3) at position c.*239A > G in the pregnant individual (II 2) of Family A, as depicted in Fig. 3 (Figure S2). Typically, locus c.*239 of Exon 8 is G in SMN1 and A in SMN2. Therefore, the presence of hybrid genes and high copy numbers may explain the asymptomatic status observed in some individuals (Fig. 3). The boy (II 2) of Family B and the mother (I 2) of Family C exhibit similar MLPA results, yet the disease manifests differently. These findings suggest the potential presence of additional factors, such as mutations, splicing modifiers, and epigenetic modifications, that may impact the severity of SMA ^28–30. Females with asymptomatic SMN1 deficiency exhibit elevated expression levels of plastin 3 (PLS3), a phenomenon that has been demonstrated to ameliorate axonal growth abnormalities in animal models of SMN1 deficiency ³¹. Therefore, it is imperative for future research endeavors to devise novel methodologies and approaches to comprehensively elucidate the genetic variations that underlie the diverse clinical manifestations of spinal muscular atrophy.

The MLPA technique is widely utilized on a global scale for detecting E7-deficient SMA. In this investigation, two false positives were detected during the MLPA analysis (Fig. 4). The occurrence of a false positive result may be attributed to the binding region of the MLPA-P060 upstream probe within the nucleotide positions c.835 − 30 to c.840 of SMN1 E7 (a total of 35 nucleotides) (https://www.mrcholland.com). If a mutation occurs within this specific region of the DNA of the individual under examination, excluding the c.840 site, the detection probe will fail to bind to the target DNA, resulting in a loss of signal. Therefore, it is crucial to meticulously analyze the MLPA-P060 results by considering various scenarios, including the presence of minor ratios or signals, the absence of a concurrent decrease in the 7/8 exon signal of SMN 1/2, and inconsistencies in the 7/8 exon sum of SMN 1/2.

Child A, aged 7 months, exhibited a lack of exertion in the lower extremities (Fig. 5). Initially, one pediatrician recommended WES for the child; however, after two weeks, a negative result was ultimately achieved. Two months after the initial examination, another pediatrician suggested the possibility of SMA in Child A. We immediately conducted MLPA and sequencing assays targeting the c.840 site to confirm the diagnosis of SMA after two days. Concurrently, the authors reviewed all WES data for Child A and identified changes in the sequencing depth of SMN1/2 E7. Child B, aged 14, was under suspicion for Duchenne muscular dystrophy (DMD) by the neurologist; however, subsequent testing using MLPA-P034/035 and WES produced negative results. Given the changes in the sequencing depth of SMN1/2 E7 in WES data, we suspected that he might have SMA, which was later confirmed by MLPA-P060.

SMA diagnosis is initially established through clinical manifestations and is confirmed by genetic analysis (Figure S3). For timely intervention for SMA families, it is imperative to promptly establish a diagnosis. Given the notable prevalence of deletions of SMN1 E7, it is recommended to prioritize the detection of deletions, particularly at the c.840 site. Our research indicates the presence of two copies of SMN1 in a patient but does not definitively rule out the possibility of 5q-SMA. Each detection method has its inherent limitations. Therefore, it is essential to integrate clinical symptoms with a comprehensive analysis of multiple detection methods. Furthermore, the diagnosis of SMA requires interdisciplinary cooperation among healthcare professionals and laboratory personnel from various departments, such as pediatrics, prenatal diagnostic centers, obstetrics, and neurology.

In conclusion, SMA presents a significant challenge for clinicians and scientists due to its extensive variability in clinical symptoms and genetic features. This study offers a comprehensive genetic analysis of suspected familial SMA cases in the Chinese population. The results enhance our understanding of the molecular genetic mechanisms underlying SMA and the utility of different molecular detection techniques, with significant implications for genetic counseling, prenatal diagnosis, and targeted therapeutic strategies in clinical practice.

SMA spinal muscular atrophy

MLPA multiplex ligation-dependent probe amplification

PCR polymerase chain reaction

WES whole-exome sequencing

IGHMBP2 immunoglobulin mu DNA binding protein

SMARD1 SMA with respiratory distress type I

CMT2S Charcot Marie Tooth type 2S

PLS3 plastin 3

DMD Duchenne muscular dystrophy

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Ethics number: KS-2018-KY-36). All participants provided voluntary informed consent before their inclusion.

Consent for publication

The authors affirm that human research participants provided informed consent for publication.

Availability of data and material

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing interests

The authors declare no conflict of interest

Funding

This work was supported by Henan Province Medical Science

and Technique Foundation (SBGJ202102164; SBGJ202102097), Key Scientific Research Projects in Colleges and Universities of Henan Province (Grant Number 22A320075), Science and Technology Huimin Project of Zhengzhou (2021KJHM0003), and the Science and Technology Research Program of Henan Province (222102520018).

Authors' contributions

GSS and KXD conceived the study. CD, LQQ, ZXC, CC, LLN, JM, ZZH and WYH helped with the implementation. All authors contributed to the refinement of the study protocol and approved the final manuscript.

Acknowledgements

We are indebted to the patients and their families for their participation in this project.

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FigureS1.tif
Figure S1. The genotype of each family member was identified as a carrier of type "2+0" or the S8Kfs*23 mutation. The letter M stands for mutation.
FigureS2.tif
Figure S2. The Sanger sequencing analysis of the pregnant individual (II 2) from Figure 3A.
FigureS3.tif
Figure S3. Flowchart for SMA Genetic Testing and Counseling.

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Editorial decision: Major revision
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Unraveling the Genetic Mysteries of Spinal Muscular Atrophy in Chinese Families

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Abstract

Objective

Methods

Results

Conclusions

Figures

Introduction

Materials and methods

Study cohort

DNA extraction

MLPA

Long-range PCR, nested PCR, and Sanger sequencing

WES

Results

An overview of the enrolled families

Fascinating discoveries during the detection

Discussion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1