Whole exome sequencing provides a diagnosis of spinal muscular atrophy pedigree with SMN1 “2+0” genotype

doi:10.21203/rs.3.rs-2130080/v1

Download PDF

Research Article

Whole exome sequencing provides a diagnosis of spinal muscular atrophy pedigree with SMN1 “2+0” genotype

https://doi.org/10.21203/rs.3.rs-2130080/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Spinal muscular atrophy (SMA) is one of the common autosomal recessive neuromuscular disease caused by mutations of the SMN1 gene. As a special SMA carrier, the “2 + 0” genotype of SMN1 poses a great challenge for carrier screening and family genetic counseling.

Methods

In this study, for the first time, we identified “2 + 0” genotype carriers via trio-based whole exome sequencing (WES) and sequencing based multiple single nucleotide polymorphisms (SNPs) haplotype linkage analysis.

Results

Combined with the copy number of SMN1 gene in family members, the genetic relationship of SMN1 pathogenic gene transmitted from grandmother to father and then to proband was confirmed.

Conclusion

Our study suggest that SNPs transmitted through multiple generations in a family could be screened by using WES sequencing to realize linkage analysis. And we determine that the SMN1 genotypes of proband’s father and grandmother are “2 + 0” genotype carriers.

Spinal muscular atrophy

multiplex ligation-dependent probe amplification

whole exome sequencing

linkage analysis

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular hereditary disease with incidence rate of 1:10,000 live births and carrier frequency of 1:60 ~ 1:35 in various populations[1]. SMA is a functional defect of motor neuron survival protein due to the mutation of SMN1 gene on 5q13.2. When this protein is defective, motor neuron cells in the anterior horn of the spinal cord undergo progressive degeneration and apoptosis. Individuals with SMA suffer from skeletal muscle atrophy, limb paralysis, respiratory failure or death[2].

In the general population, there can be 0 ~ 4 copies of SMN1 on each chromosome 5. Usually, normal individuals carry one copy of SMN1 on each chromosome, which is “1 + 1” genotype. About 95% of SMA patients are caused by homozygous deletion of SMN1 with “0 + 0” genotype[3]. Most SMA carriers with “1 + 0” that carry a chromosome with 1 copy of SMN1, while SMN1 is deleted on the other chromosome. However, in 3.3 ~ 8.5% of individuals[4], there are two copies of SMN1 in cis state on one chromosome, while there is no copy on the other chromosome. Although the total copy number of SMN1 in these individuals is 2, they are still SMA carriers. And this genotype is called “2 + 0”. Common techniques for detecting SMN1 copy number include multiplex ligation-dependent probe amplification (MLPA), quantitative real-time polymerase chain reaction (qPCR), denaturing high-performance liquid chromatography (DHPLC) etc. All of these techniques can not effectively detect SMN1 “2 + 0” genotype[5, 6]. It needs to be combined with linkage analysis to make a clear diagnosis.

Single nucleotide polymorphism (SNP) mainly refers to the DNA sequence polymorphism caused by single nucleotide variation at the genomic level, accounting for about 90% of human DNA polymorphism. SNPs can be used as a marker of chromosome origin analysis, which is called the third generation genetic marker. In this study, we performed whole exome sequencing (WES) on a SMA family suspected of “2 + 0” genotype, screened out SNPs loci for linkage analysis, and combined with MLPA to determine the distribution of SMN1 in this family.

Patient

The proband is an 8-month-old girl born at 40 weeks of gestation. The mother was G1P1 and the perinatal period was normal. She was weak in standing and sitting and presented to the Third Affiliated Hospital of Zhengzhou University for treatment of delayed motor development. During the physical examination, the doctor found that the proband's lower limbs were weak and could not support her weight, and the knee tendon reflex of both lower limbs was not elicited. Cardiac function test showed that creatine kinase increased slightly, electromyography showed extensive neurogenic damage in the limbs, and the preliminary clinical diagnosis was SMA. The proband's parents, grandparents and uncle are health, denying the history of next-of-kin marriage. Patient of her family members signed informed consent, and the study passed the ethical approval of Third Affiliated Hospital of Zhengzhou University.

Specimen Collection And Dna Extraction

Genomic DNA was obtained from the peripheral blood specimens of proband, parents, grandparents and uncle. Using Qiagen DNA Blood Midi/Mini kit (Qiagen GmbH, Hilden, Germany) for Genomic DNA Extraction, following the manufacturer’s protocol.

Copy number analysis of SMN1

SMN1 copy numbers were determined by MLPA using the probemix P060 SMA kit (MRC-Holland, Amsterdam, Netherlands). 50ng genomic DNA was denatured for probe hybridization, ligation and PCR reaction. The PCR products were subjected to capillary electrophoresis with ABI 3500dx sequencer (Applied Biosystems, Carlsbad, USA). The MLPA data were analyzed using the Coffalyser software package (MRC-

Holland®).

Whole Exome Sequencing

50 ng genomic DNA was interrupted to 200bp around by fragmentation enzymes. The DNA fragments were then end repaired, ligated with barcoded sequencing adaptors and collected by XP beads. After PCR amplification, the DNA fragments were hybridized and captured by NanoWES (Berry Genomics, Beijing, China) according to the manufacturer’s Protocol. Novaseq6000 platform (Illumina, San Diego, USA) was used for sequencing the genomic DNA of the family.

Data Analysis

The sequencing reads were aligned to the human reference genome (hg19/GRCh37) using Burrows–Wheeler Aligner tool and PCR duplicates were removed by using Picard v1.57 (http://picard.sourceforge.net/). Using GeneRanger-Haplotype analysis software (Xunyin Biotechnology Co., Shanghai, China), the family SNPs and haplotype were analyzed.

Linkage Analysis

Previous studies had found that[7, 8], linkage analysis of SMA families could be realized by using four STR loci. Through the comparison of forensic kinship identification data, the comprehensive analysis of 3 ~ 4 SNPs was equivalent to the detection result of 1 STR locus[9, 10]. Therefore, according to the results of family haplotype analysis and taking the SMN1 (NC_000005.9:g.(70220768..70248842)) as the target region, we selected at least 12 heterozygosity loci in SMN1 upstream and downstream as effective SNPs. Linkage analysis was carried out to clarify the genetic origin of chromosomes and the distribution of SMN1 genes.

Copy number variations of SMN1

The diagnostic analysis workflow of SMN1 copy number variations of the family was summarized in Fig. 1. The proband carried SMN1 homozygous deletions and diagnosed as SMA, combined with clinical phenotype. The mother had 1 copy of SMN1, so she was a SMA carrier. The father had 2 copies of SMN1 and his clinical phenotype is normal. So the father was suspected to be with “2 + 0” genotype. Further MLPA analysis of the grandparents and uncle, we were found that the grandmother was also suspected to be with “2 + 0” genotype. Results of MLPA analysis were in the Supplementary figure.

Linkage analysis and distribution of SMN1 copy number in family

Based on the WES data of the third generation members in the family (Supplementary tables), 12 heterozygosity SNP loci of grandmother and 15 heterozygosity SNP loci of grandfather were screened within the upstream and downstream 6Mb of SMN1. Linkage analysis of these SNP loci was carried out to clarify the genetic pattern of chromosomes in families.

The results of SNPs linkage analysis were shown in Table 1 and Table 2. Father and uncle inherited different chromatids from the grandmother, that was, different maternal chromosomes. On the other hand, father and uncle had the same origin of paternal chromosomes. Combined with the SMN1 copies of the proband and the uncle, it was determined that the father carried 2 copies of SMN1 from the grandfather, while the other allele with 0 copy was inherited from the grandmother and passed on to the proband through the father (Fig. 2). At the same time, the mother passed 0 copy of SMN1 to the proband, resulting in SMA in the child.

The copy of SMN1 of proband’s grandfather, grandmother and uncle were 3, 2 and 4, respectively. Combined with the linkage analysis of SNPs, it was suggested that the SMN1 genotype of uncle was “2 + 2” and inherited from proband’s grandfather and grandmother, respectively. Grandfather was “2 + 1” genotype, so it was basically determined that both father and grandmother are “2 + 0” genotype. Moreover, through the copy number analysis of SMN1 and SNPs linkage analysis, the denovo deletion of SMN1 in the proband was basically excluded.

SMA is a kind of neuromuscular disease with high carrier rate and high morbidity, which can lead to disability or death. Both American College of Medical Genetics and Genomics (ACMG)[11] and American College of Obstetricians and Gynecologists (ACOG)[12] recommend screening for SMA should be offered to all women who are considering pregnancy or are currently pregnant.

Among Ashkenazi Jewish (AJ) individuals, the detectability of SMA carrier is about 90%. Of the remaining 10%, ~ 8% are “2 + 0” genotype carriers[4]. Verhaart et al.[13], combined with published epidemiological data, showed that the carrier rate of “2 + 0” in the general population is about 5% ~ 8%. However, the current techniques for SMN1 copy detection can not effectively distinguish SMN1 “2 + 0” from “1 + 1” normal genotype, and it needs to be combined with linkage analysis to confirm the diagnosis.

As the third generation genetic marker, SNPs analysis has the following characteristics: 1) There is a large number and high density of SNPs, with an average of one SNP per 1000 base pairs in the human genome. 2) SNPs are highly conservative. The average mutation rate of SNPs is about 1.8×10^− 8 [14], lower than short tandem repeat (STR).

Due to the wide distribution of SNPs, WES capture chip can detect a certain number of SNPs sites effectively. Moreover, compared with the expensive genome-wide SNP arrays, WES is more suitable for SNPs detection and linkage analysis of single gene hereditary diseases.

It is generally believed that the closer SNPs are to the target gene, the stronger the linkage between SNPs and the target gene. Chong et al.[15] performed whole-genome SNP arrays on 1,415 Hutterites, and found no SNPs were present within 2Mb at the SMN1 locus. In this study, we analyzed WES data of a single core family. A total of 13 effective SNPs were screened within 2Mb of SMN1, so the linkage analysis obtained is more relevant.

Through MLPA and SNPs linkage analysis, it indicated that the proband's grandfather inherited the chromosome monomer with two SMN1 copies to her father and uncle. And the grandmother passed on the 0 copy of chromosome to the proband through her father. Thus, it was concluded that both the grandmother and father were “2 + 0” genotype. The results suggested SNPs that transmitted through multiple generations in a family could be screened by using WES sequencing data to realize linkage analysis. At the same time, the capture chip used by WES is universal, so it is no longer necessary to design primers for SNPs for a specific disease.

In addition, with the rapid decline of the cost of high-throughput sequencing, WES has been widely used in the clinical diagnosis of genetic diseases in children. Several studies have recommended WES as the first-line diagnostic test for patients with undiagnosed diseases or neurodevelopmental disorders[16–18]. WES also be used as a carrier screening technique for autosomal recessive and X-linked diseases in the field of reproductive medicine[11]. So using WES data to linkage analysis can also meet the needs of family genetic disease diagnosis and carrier screening.

In general, this study used WES to screen the SNPs of three generations in a SMA family. Combined SMN1 copy number and linkage analyses indicated that both proband’s father and mother had two SMN1 copies on one allele, confirming the “2 + 0” genotype. On the other hand, trio-WES analysis could also meet the needs of the family for screening carriers of common genetic diseases, provide more accurate and comprehensive disease diagnosis and genetic counseling for patients, and provide more methods for the detection of carriers with suspected SMN1 genotype “2 + 0”.

Ethics Approval and Consent to Participate

The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013) and was approved by the Ethics Committee of The Third Affiliated Hospital of Zhengzhou University (Approval No. 2020-057-01). Written informed consent to patient was obtained from the parents of the patient, who was under the age of 16.

Consent for Publication

Written informed consent was obtained from the patient and her parents to publish this case report and any accompanying dates. And the manuscript is approved by all authors for publication.

Availability of data and materials

All data generated or analysed in this study are included in this published article and its Supplementary Materials. The raw datasets used and analysed during the current study are not deposited in publicly available repositories because of considerations about the security of human genetic resources. For other details of the availability of data and material, please refer to the methods section of the article and tables. Meanwhile, sequencing dataset can be obtained from any of the two corresponding authors on reasonable request.

Competing interests

The authors declare that there are no financial or other relationships that might lead to a conflict of interest of the present article.

Funding

This study was approved by Medical Science and Technique Foundation of Henan Province (LHGJ20200453 and LHGJ20200462).

Acknowledgement

We thank the patient and her families who kindly agreed to participate in this study.

Author’s Contribution

All authors have materially participated in the study and manuscript preparation. Xiaoli Zhang and Jinshuang Gao collected all clinical data, performed the clinical and genetic analyses and drafted the manuscript; Yaqing Guo and Xiao Li analysed the genetic data; Chenyu Zhang and Mengyue Wang performed laboratory experiments; Haiyang Yu and Erfeng Yuan participated in the design of some experiments and conducted the in silico analysis of data; Xiaoli Li, Hao Chen and Jing Guan provided patient care and assessment for motor function; Tianming Jia provided patient care, neurology expertise and participated in the design of study; Linlin Zhang designed the whole study and drafted and revised the manuscript. All authors have approved the final article.

Alías L, Bernal S, Calucho M, Martínez E, March F, Gallano P, et al. Utility of two SMN1 variants to improve spinal muscular atrophy carrier diagnosis and genetic counselling. European journal of human genetics : EJHG. 2018;26(10):1554-57.
Mercuri E, Finkel RS, Muntoni F, Wirth B, Montes J, Main M, et al. Diagnosis and management of spinal muscular atrophy: Part 1: Recommendations for diagnosis, rehabilitation, orthopedic and nutritional care. Neuromuscular disorders : NMD. 2018;28(2):103-15.
Cao Y, Cheng M, Qu Y, Bai J, Peng X, Ge X, et al. Factors associated with delayed diagnosis of spinal muscular atrophy in China and changes in diagnostic delay. Neuromuscular disorders : NMD. 2021;31(6):519-27.
Luo M, Liu L, Peter I, Zhu J, Scott SA, Zhao G, et al. An Ashkenazi Jewish SMN1 haplotype specific to duplication alleles improves pan-ethnic carrier screening for spinal muscular atrophy. Genetics in medicine : official journal of the American College of Medical Genetics. 2014;16(2):149-56.
Wu J, Lu AD, Zhang LP, Zuo YX, Jia YP. [Study of clinical outcome and prognosis in pediatric core binding factor-acute myeloid leukemia]. Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi. 2019;40(1):52-57.
Su YN, Hung CC, Lin SY, Chen FY, Chern JP, Tsai C, et al. Carrier screening for spinal muscular atrophy (SMA) in 107,611 pregnant women during the period 2005-2009: a prospective population-based cohort study. PloS one. 2011;6(2):e17067.
Yanyan C, Miaomiao C, Fang S, Yujin Q, Jinli B, Hong W. Familial study of spinal muscular atrophy carriers with SMN1 (2+0) genotype. Yi chuan = Hereditas. 2021;43(2):160-68.
Sharifi Z, Taheri M, Fallah MS, Abiri M, Golnabi F, Bagherian H, et al. Comprehensive Mutation Analysis and Report of 12 Novel Mutations in a Cohort of Patients with Spinal Muscular Atrophy in Iran. Journal of molecular neuroscience : MN. 2021;71(11):2281-98.
Gill P. An assessment of the utility of single nucleotide polymorphisms (SNPs) for forensic purposes. International journal of legal medicine. 2001;114(4-5):204-10.
Budowle B, van Daal A. Forensically relevant SNP classes. BioTechniques. 2008;44(5):603-8, 10.
Gregg AR, Aarabi M, Klugman S, Leach NT, Bashford MT, Goldwaser T, et al. Screening for autosomal recessive and X-linked conditions during pregnancy and preconception: a practice resource of the American College of Medical Genetics and Genomics (ACMG). Genetics in medicine : official journal of the American College of Medical Genetics. 2021;23(10):1793-806.
Committee Opinion No. 691 Summary: Carrier Screening for Genetic Conditions. Obstetrics and gynecology. 2017;129(3):597-99.
Verhaart IEC, Robertson A, Wilson IJ, Aartsma-Rus A, Cameron S, Jones CC, et al. Prevalence, incidence and carrier frequency of 5q-linked spinal muscular atrophy - a literature review. Orphanet journal of rare diseases. 2017;12(1):124.
Kondrashov AS. Direct estimates of human per nucleotide mutation rates at 20 loci causing Mendelian diseases. Human mutation. 2003;21(1):12-27.
Chong JX, Oktay AA, Dai Z, Swoboda KJ, Prior TW, Ober C. A common spinal muscular atrophy deletion mutation is present on a single founder haplotype in the US Hutterites. European journal of human genetics : EJHG. 2011;19(10):1045-51.
Manickam K, McClain MR, Demmer LA, Biswas S, Kearney HM, Malinowski J, et al. Exome and genome sequencing for pediatric patients with congenital anomalies or intellectual disability: an evidence-based clinical guideline of the American College of Medical Genetics and Genomics (ACMG). Genetics in medicine : official journal of the American College of Medical Genetics. 2021;23(11):2029-37.
Srivastava S, Love-Nichols JA, Dies KA, Ledbetter DH, Martin CL, Chung WK, et al. Meta-analysis and multidisciplinary consensus statement: exome sequencing is a first-tier clinical diagnostic test for individuals with neurodevelopmental disorders. Genetics in medicine : official journal of the American College of Medical Genetics. 2019;21(11):2413-21.
Trujillano D, Bertoli-Avella AM, Kumar Kandaswamy K, Weiss ME, Köster J, Marais A, et al. Clinical exome sequencing: results from 2819 samples reflecting 1000 families. European journal of human genetics : EJHG. 2017;25(2):176-82.

Tables 1 and 2 are available in the Supplementary Files section.

SupplementaryMaterials.rar
Supplementary figure 1: The copies of SMN in the family; Supplementary table 1: Haplotype analysis of chromosome 5 based on WES sequencing data of the proband and parents; Supplementary table 2: Haplotype analysis of chromosome 5 based on WES sequencing data of the proband's parents and grandparents; Supplementary table 3: Haplotype analysis of chromosome 5 based on WES sequencing data of the proband's uncle and grandparents.
Tables.xlsx

Download PDF

Version 1

posted

You are reading this latest preprint version

Whole exome sequencing provides a diagnosis of spinal muscular atrophy pedigree with SMN1 “2+0” genotype

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

Patient

Specimen Collection And Dna Extraction

Whole Exome Sequencing

Data Analysis

Linkage Analysis

Results

Discussion

Declarations

Ethics Approval and Consent to Participate

Consent for Publication

Availability of data and materials

Competing interests

Funding

Acknowledgement

Author’s Contribution

References

Tables

Supplementary Files

Status:

Version 1