Carrier Screening of SMA in North China

Background: Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by loss of motor neurons and progressive proximal muscular atrophy. Although the SMN1 carrier frequencies were reported between 1:53- 1:83 in the Chinese population, carrier frequencies for many ethnicities, including most ethnic groups in North China, are unknown. Methods: A total of 3,130 maternal blood samples were collected at the Lvliang People’s hospital and Tonghua Central hospital. This research was performed with a three-stage screening procedure. The pregnant women were rst examined for exon 7 and exon 8 copy number of SMN1. If the woman was determined as a SMA carrier, her partner was also tested, and if both parents were carriers, prenatal diagnosis was recommended. The copy number of both exons 7 and 8 of SMN1 gene were identied by quantitative real-time polymerase chain reaction according to the manufacture’s instructions. Results: A total of 3,130 pregnant women including 1,405 cases from Lvliang People's Hospital and 1725 cases from Tonghua Central Hospital, were tested for SMA carriers using real-time PCR assay. Seventy six cases were heterozygous deletion of exons 7 and 8 in SMN1 gene [1 + 0 genotype], thus carrier frequency of SMN1 deletion is 1:42 (2.43%). After detailed genetic counseling, 52 related paternal partners were tested. Among those individuals, a couple from Tonghua was found to be high risk for having offspring with SMA and prenatal diagnosis was then implemented, and the fetus was diagnosed with SMA. The carrier of SMA frequency in Lvliang and Tonghua populations were 1: 57 and 1: 34 respectively. Therefore, the carrier frequency in Lvliang (1:56) was signicantly lower than that in Tonghua (1:34) (p=0.0330). The prevalence of SMA in Lvliang and Tonghua populations were estimate to be 8.5E-5 and 2.25E-4 respectively. Conclusion: In conclusion, our research has determined the distribution of SMA carrier frequency in the general pregnancies that are present in the northern of China population. This study also provides an accurate assessment of allele frequencies and estimates of SMA prevalence that were previously unavailable to clinicians and patients considering testing in the north of China.


Introduction
Spinal muscular atrophy (SMA) is one of the most common autosomal recessive neuromuscular disorders affecting infants and children, characterized by degeneration of motor neurons and progressive muscular atrophy [1]. The incidence of all types of SMA is estimated to be 1 in every 10,000 live births globally [2]. SMA is caused by mutations in SMN1 gene on chromosome 5q13.2. 95% of SMA patients have homozygous SMN1 deletions, 5% of SMA patients have compound heterozygous SMN1 deletion and point variant. Most patients inherit variants from their parents. Only approximately 2% of SMA patients have de-novo variants [3]. SMA is categorized into ve types, which are based on motor function obtained and age of onset. Type 0 and I are the most extreme types with SMA onset beginning at birth. Patients usually achieve minimal motor function and develop respiratory failure, their life expectancy is around six months to two years [4]. Type II and III are the intermediate forms of the disorder where affected children are able to sit or walk for a short distance. Depending on their level of the weakness respiratory muscle, the survival time of patients may be beyond of 10 years of age. Type IV SMA begins during adult where patients have a normal life expectancy [5]. Up today,the US Food and Drug Administration approved NUSINERSEN (in December 2016) and ZOLGENSMA (in May 2019) for treatment SMA patients. NUSINERSEN was the rst drug approved to treat pediatric and adult patients with SMA while ZOLGENSMA was the rst gene therapy approved to treat SMA children under two years old. These e cacies of novel drugs have recently been demonstrated that improve or ameliorate symptoms in many SMA patients [6]. With the availability of treatment options, the knowledge of carrier frequencies of SMN1 is essential for prevention or early treatment for this disease [7]. Although the SMN1 carrier frequencies were reported between 1:53 − 1:83 in the Chinese population, most of which were studied in south of China [8,9]. The SMA carrier frequencies may be variable in different geography in China and the information on the prevalence of SMA of the North China is limited, especially in Lvliang (the northwest) and Tonghua (the northeast). Therefore, we conducted a comparative study to estimate the SMN1 allele frequency in  Table 2. Therefore, the carrier frequency in Lvliang and Tonghua were estimated to be 1:56 and 1:34 respectively. All of the SMA carrier pregnancies were given detailed genetic counseling regarding the etiological factors, inheritance pattern, clinical features, reproductive risk and the treatment of SMA. After being made fully aware of the disease, 14/25 (56%) male partners in Lvliang and 38/51 (74.51%) male partners in Tonghua were also recalled and tested for SMN1 copy number by qPCR, of whom 1 partner (1/38, 2.63%) in Tonghua was found to be a SMA carrier (Table 1). One couple of high risk was offered invasive prenatal diagnosis of SMA. The exon 7 and exon 8 copy number of SMN1 and SMN2 genes of amniotic uid sample were determined by CNVplex®. The results of amniocentesis indicated that this woman was carrying a fetus with homozygous deletion of SMN1 gene and three copies in exon 7 of SMN2. The detailed copy numbers of SMN1 and SMN2 of patients were shown in Table 2. Previous studies have reported that SMA phenotype is mostly caused by homozygous deletions [11,12]. After careful prenatal counseling, the pregnancy was terminated, and the fetus tissue then was also con rmed to have SMA with homozygous deletion of the exon 7 and 8 of the SMN1 gene. Results of this family were shown in Table   3 and Fig. 2. Additionally, the two copy and three copy genotype frequencies of SMN1detected in our data are 96.39% and 1.82% respectively. The 96.39% of two copies frequencies is higher than the 92.55% has been reported in the Chinese population (3.84%, p = 0.0001). However, the 1.82% three copy genotype frequencies of SMN1 in this research is lower than the 5.68% that reported in the Chinese population (3.86%, p = 0.0001) [13].  Hardy-Weinberg equilibrium was used to calculate the frequencies of SMN1 copies per allele  (Table 5). These calculations reveal that the expected the prevalent of SMA in the Lvliang and Tonghua group is 8.5E-5 and 2.25E-4 respectively. The prevalent of SMA in Tonghua population is comparable to the Russian populations that reported in previous study (p = 0.9979) (* Table 6). Our study had some limitations. Due to the limitation of qPCR method, 5% of SMA patients with SMN1 missense variant and silent carrier genotype SMN1 '2 + 0' could not be identi ed by the qPCR. Therefore, the SMA carrier frequency might be underestimated than that could be. By contrast, this study also had several advantages. This is the rst large-scale population study on the SMA carrier frequency in Northern of China conducted using samples from pregnant women. Further, this study also provides an accurate assessment of allele frequencies and estimates of SMA prevalence that were previously unavailable to clinicians and patients considering testing in the north of China.

Conclusion
In conclusion, our research has indicated SMA carrier frequency in the northern of China. The results also gave a valuable data for a nationwide program of genetic counseling, population screening and clinical/prenatal diagnosis to prevent SMA in China. Such an approach should provide Chinese couples who are undergoing genetic counseling with improved choices for their family planning. procedure. At the rst stage, the pregnant women were examined for exon 7 and exon 8 copy number of SMN1. If a woman was found to be a SMA carrier, her partner was also recommended for testing, and if both parents were carriers, prenatal diagnosis was recommended.
All of the screening participants were given standard genetic counseling. Information on the participants was listed in Fig. 1 and Table 2.

SMA screening test
A total of 5 mL of peripheral blood was collected from each participant. Genomic DNA was extracted using GO- . The ampli cation of SMN1 and RPP40 genes (internal standard) were done by multiplex PCR. Each assay included ve control samples, which were a no-DNA control, one SMN1 exon 7 or exon 8 deletion control, and three gradients control of two-copy of SMN1. All samples were performed by an ABI StepOne plus real-time PCR system. The data from FAM and VIC channels were used to calculate the orescence and evaluate the cycle threshold (Ct). ΔCt and ΔΔCt were then calculated to de ne the SMN1 copy number by using the 2 −ΔΔCt method (Table 7). The copy number of exon 7 and exon 8 of SMN1 gene were calculated according to scheme shown in the Table 1.

Clinical follow-up
Pregnancies with wild type SMA screening results were advised for regular prenatal care; genetic counseling was provided if routine ultrasound examination showed abnormalities. Pregnant women with heterozygous deletion SMN1 results were given detailed genetic counseling regarding the etiological factors, inheritance pattern, clinical features, reproductive risk and the treatment of SMA and her partners were offered SMA screening test. If both partners were de ned as SMA carriers, the genetic counseling was provided for invasive prenatal genetic diagnostic testing of the fetus. The copy number of SMN1 and SMN2 genes of amniotic uid sample were explored by CNVplex® (a technique for high-throughput detection of sub-chromosomal copy number aberrations) as described before [10]. The homozygous deletion SMN1 of fetus result was de ned as true positive upon postnatal genetic diagnostic con rmation or clinical follow-up results. Patients without con rmatory diagnostic results were excluded from this research.

Statistical analysis
Statistical analysis between the different groups was performed using a chi-square test or Fisher's exact test, and P values of < 0.05 were considered statistically signi cant.