Volume 185, Issue 1 pp. 274-277
RESEARCH LETTER
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Carrier frequency of SMN1-related spinal muscular atrophy in north Indian population: The need for population based screening program

Mayank Nilay

Mayank Nilay

Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

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Amita Moirangthem

Amita Moirangthem

Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

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Deepti Saxena

Deepti Saxena

Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

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Kausik Mandal

Kausik Mandal

Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

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Shubha R. Phadke

Corresponding Author

Shubha R. Phadke

Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

Correspondence

Shubha R. Phadke, Department of Medical genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow, 226014, UP, India.

Email: [email protected]

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First published: 14 October 2020
Citations: 17

Abstract

Chromosome 5q related Spinal muscular atrophy (SMA) is an autosomal recessive, progressive, neuromuscular disorder most commonly caused by homozygous deletion of exon 7 or exon 7 and 8 of SMN1 gene. Being the leading genetic cause of infant mortality, studies of its prevalence and incidence are necessary. Carrier testing for the common pathogenic variant for SMA is offered to the couples visiting our tertiary care hospital in North India. Subjects were tested for SMA carrier status by Multiplex Ligation-dependent Probe amplification (MLPA) technique for deletion of exons 7 and 8 of SMN1 gene. The retrospective data of individuals tested for SMA carrier status in last 4 years (2016–2019) was evaluated. Six hundred and six individuals without family history of SMA or carrier of SMA who were subjected to MLPA based screening for SMA carrier status were included in the study. The carrier frequency of SMN1 deletion (deletion of exon 7 and/or exon 8) was found to be 1 in 38 (16 out of 606). The catchment area of our medical genetics clinic covering the state of Uttar Pradesh (16.5% of Indian population according to censusindia.gov.in, 2011) and neighboring states, showing SMA carrier frequency of 1:38 in a cohort with no prior positive family history has important significance for policy making.

1 INTRODUCTION

Spinal muscular atrophy (SMA) is characterized by motor neuron degeneration in the anterior horn cells and consequent muscle atrophy leading to debility or mortality. SMA presentation varies in relation to onset and severity with SMA type I (age of onset <6 months) being the commonest and severest form. Median age of survival varies between 8 and 13.5 months (Finkel et al., 2014; Kolb et al., 2017). It is inherited in an autosomal recessive manner with 25% chance of occurrence in each pregnancy if both the partners are carriers. Autosomal dominant and X-linked spinal muscular atrophy is also seen rarely (Ibrahim et al., 2012). Ninety six percent of cases of SMA are caused by pathogenic variants in SMN1 gene located on 5q11.2-13.3 (Wirth, 2000). The commonest causative variant is deletion of exons 7 and 8 or exon 7 only, accounting for most of the cases of SMA. The deletion present in homozygous form accounts for 96.4% cases of 5q related SMA (Wirth, 2000). In the rest, one copy of SMN1 has this exonic deletion while the other allele carries a pathogenic sequence variation. SMA due to solitary deletion of exon 8 of SMN1 gene has also been reported (Gambardella et al., 1998). The estimated prevalence of SMA is approximately 1–2 per 100,000 persons and incidence is around one in 10,000 live births (Verhaart et al., 2017). Various studies on population carrier frequency of SMA have been done in western countries and other small geographical regions involving various ethnicities but such studies are lacking in South East Asian populations. Because of severity of the disorder, the prevention by population based screening is justified. With the availability of drugs like Nusinersen, Risdiplam and Onasemnogene abeparvovec, newborn screening and early treatment initiation can be another viable option. We offer carrier testing for SMA as well as some other common genetic conditions like beta thalassemia and fragile X syndrome to the couples visiting our medical genetics clinic for preconceptional counseling or in early pregnancy. This study is an attempt to calculate the carrier frequency of SMA from this cohort.

2 METHODS

We retrospectively analyzed the records for SMA carrier status done at our centre in last 4 years (2016–2019). All couples or women coming for consultation prepregnancy or in early pregnancy are offered screening for SMA with adequate counseling about the disorder. These women or couples are usually seen for the history of spontaneous abortions; poor reproductive outcomes like intrauterine fetal demise or neonatal death; previous child with history of malformation, intellectual disability or some genetic disorder; exposure to teratogenic agent or infrequently routine prepregnancy counseling. Either husband or wife of child bearing age who opted-in for carrier testing after proper genetic counseling was tested by Multiplex Ligation-dependent Probe Amplification (MLPA) technique. This study was approved by institutional ethics committee with waiver of consent. Those individuals who were tested due to prior family history of SMA or any childhood death with suspected hypotonia were excluded from this observational study. Genomic DNA was extracted from the blood leucocytes using standard procedures of QIAamp DNA blood mini kit (Qiagen Inc.). DNA concentration and quality were checked with the NanoDrop 8000 spectrophotometer. A concentration of 50 ng/μL of DNA was used for each analysis.

MLPA was carried out as per manufacturer's instructions (SALSA MLPA probemix P060 SMA Carrier, MRC-Holland, Amsterdam, The Netherlands). This probe mix has 21 MLPA probes with two probes each for SMN1, SMN2 genes, and 17 reference probes that detects sequences outside this region. Data were analyzed using Coffalyser.Net software. A dosage quotient (DQ) of the probe between 0.40 and 0.65 (i.e., 0.40 < DQ < 0.65) represents heterozygous deletion (copy number 1) whereas DQ between 1.30 and 1.65 signifies heterozygous duplication (copy number 3). A normal (i.e., allele copy number 2) is depicted by a DQ between 0.80 and 1.20.

3 RESULTS

Six hundred and twenty six individuals were tested for carrier status (Table 1). Twenty of them with prior family history suggestive of SMA were excluded. Thus, 606 individuals, that is, 323 males and 283 females fulfilled the eligibility criteria and formed the study set. None of them were relatives of known SMA carrier. In most of the selected cases, one member of the couple was tested. In some nonconsanguineous couples who presented especially during first trimester of pregnancy, both the partners were tested for SMA carrier status simultaneously. Sixteen individuals were detected to have a positive carrier status for SMA that is, heterozygous deletion in exon 7 of SMN1 (two), heterozygous deletion in exon 8 of SMN1 (one) or both exon 7 and exon 8 of SMN1 gene (13). The partners of these 16 individuals when tested were not found to be carriers. These 16 partners as a part of second level testing are not included for the calculation of carrier frequency. Thus, the carrier frequency in our selected cohort was found to be 1:38 (16 out of 606; Table 2). Applying the Hardy–Weinberg principle with this carrier frequency (one in 38), the incidence of SMA comes around one in 5620 live births. The laboratory provided prenatal diagnosis to 49 pregnancies for previous child with SMA. The fetuses affected with SMA were 10 (20%) which is close to the expected figure of 25%. The number of fetuses who were carriers for SMA was 17 (~35%).

TABLE 1. Results of the study in tabular form
Parameters Number(n)
Total subjects tested for carrier status 626
Male 334 (53.4%)
Female 292 (46.6%)
Median age in years (female) 29
Median age in years (male) 32
Carriers with positive family history (A)

20 (Male-11, Female-09)

Consanguinity-03

Carriers with no positive family history (B)

16 (Male-07, Female-09)

Consanguinity-01

Carrier frequency (excluding A) 1 in 38
Prenatal tests done (positive family history) 49
Fetus affected 10
Fetus unaffected 39 (carriers-17)
  • a Carriers with positive family history were not included for calculation of carrier frequency.
TABLE 2. Comparison with previous studies on Indian ethnicity
Reference Country/cohort Ethnicity No. of carriers Total no. 1 in Carrier frequency 95% C.I.
Sugarman et al. 2012 USA/Puerto Rico Asian Indian 17 976 52 0.0174 (0.010, 0.028)
Larson et al. 2015

1000 Genomes Project

(phase 3)

Asian Indian 03 489 163 0.0061 (0.001, 0.018)
Our study India North Indian 16 606 38 0.0264 (0.015, 0.043)
  • a 95% C.I. (Confidence Interval) calculated using Binomial “exact” estimation method.
  • b Carrier frequencies calculated by authors considering “2 + 0”, “1 + 1D” and “2 + 1D” genotypes into account and thus adjusting for false negatives. “D” represents defective gene due to point mutation or microdeletion on other chromosome.
  • c Carrier probability determined using DNA-sequencing based computational protocol and also corroborated with MLPA test.

4 DISCUSSION

The American College of Medical Genetics (ACMG) and the American College of Obstetricians and Gynecologists (ACOG) recommend universal screening for SMA carrier status because of clinically severe presentation and a high carrier frequency (Prior, 2008; Rink et al., 2017). The carrier frequency in the small group of 606 Indians is 1 in 38 or 0.0264 (95% Confidence interval: 0.015–0.043) in this study. Though there is general impression that SMA is one of the common genetic disorders in India, there is no data on incidence or prevalence of this disease in this country. The available data in published literature has shown carrier frequency of one in 52 in Asian Indians (Table 2; Sugarman et al., 2012). This is a large population based study (excluding those with positive family history) of carrier frequency for SMA as seen in six major ethnic groups in the United States. The carrier rates varied from 1 in 47 in the Caucasian population to 1 in 72 in the African Americans. Larson et al. (2015) applied a Bayesian hierarchical model to measure an individual's carrier probability from the exome data across multiple ethnicities (1000 Genomes dataset) and found a carrier frequency of 0.006 in the Asian Indian cohort (Table 2). However, these carrier frequencies basically represent Americans of Indian descent, which does not necessarily signify the carrier status in the subcontinent. About 94% of screening population comprised of females in the pan-ethnic cohort tested by Sugarman et al. (2012) while in our small cohort we observed an active participation (53%) of the male partners in couple opting in for carrier testing. While offering the test, we provided an option of obtaining blood sample from a single partner. As females provide various samples for other investigations for the primary reason of referral like recurrent spontaneous abortions and fragile X screening, we suggested obtaining the blood sample from the male partner for beta thalassemia and SMA screening. This is usually well received by them as shown in this data.

The carrier frequency in Caucasians and Ashkenazi Jews is found to be higher than Hispanics, Asians, and African American populations (Hendrickson et al., 2009). Higher carrier frequencies are reported in Morocco (1 in 25), Iran and Saudi Arabia (1 in 20) probably due to high level of consanguinity while European populations have shown carrier rates ranging from 1 in 50 to 1 in 80 (Lyahyai et al., 2012). Though a large number of cases of SMA (suspected and diagnosed) are seen in India, there is no planned population based screening program for SMA. Some medical genetics centres have started recommending screening for SMA and fragile X along with beta thalassemia. Due to one common pathogenic variant accounting for most of the cases, population based screening is feasible. MLPA is an easy and relatively cheap technique and can detect deletions in SMN1 gene. However, it will miss carriers with pathogenic point mutations and a rare type of situation where one chromosome has deletion while the other chromosome has two copies of SMN1 gene (2 + 0) (Calì et al., 2014). Currently the molecular techniques to identify “2 + 0” genotype are tedious and indirect. Luo et al. (2014) had used linked haplotypes which may vary from population to population. The data on contribution of these rare causative variants for SMA in India is not available though their existence is supported by family studies of rare cases of SMA in whom only one of the parents was found to be a carrier of SMN1 gene deletion. The frequency of this 2 + 0 genotype varies in different populations (African American group—27% compared to 3.3–8.1% in other ethnicities) (Hendrickson et al., 2009). Also de novo deletion of exon 7 of one SMN1 allele occurs in 2% of individuals with SMA; where only one parent is a carrier and risk of recurrence in future pregnancies is low. MLPA or real-time qPCR will remain the main techniques for detecting carriers of SMA with a caveat of missing carriers with 2 + 0 genotype until the availability of better methods. These limitations need to be conveyed to families who are reported to be a non-carrier by MLPA testing to avoid the rare possibility of misdiagnosis.

With availability of drugs like Nusinersen, Onasemnogene and Risdiplam with good results, neonatal screening is also a viable option. A few SMA patients in India have gained access to these drugs through humanitarian access programs in coordination with respective pharmaceutical companies and SMA support groups. The government is taking steps for making policies for supporting treatments for these rare genetic disorders. Currently due to cost of the treatment, an option of antenatal screening and prenatal diagnosis remains an important measure to tackle the burden of a disease with poor outcome.

We found a SMA carrier rate of 1 in 38 (2.64%) based on a sample of 606 nonrelated healthy individuals with no prior family history of SMA. This figure is comparable with that of other common disorders like β thalassemia (average prevalence of carriers is 3–4%) (Colah et al., 2017) for which population based carrier screening is advocated and is widely accepted in India. Thalassemia screening due to pilot programs of population based screening supported by government and non-government organizations over the last three decades have achieved huge success and many obstetricians are offering beta thalassemia screening during early pregnancy. The multicentre Jai Vigyan programme of the Indian Council of Medical Research on community control of thalassemia strengthened centers in different states for screening programmes and also helped in determining the prevalence of β thalassemia. These programmes had a considerable impact and pan India Thalassemia screening is being undertaken in different target groups along with prenatal testing (Colah et al., 2017). Prenatal testing with an option of termination of affected fetus is accepted by most of the population groups in India.

An important limitation in this study was the biased selection as cohort consisted of many subjects seeking medical intervention due to prior family history of some adverse events. The carrier frequency detected in our cohort with predicted incidence of 1 in 5620 live births represents significant disease burden. This study on the carrier status of SMA in north Indian population will therefore be a stepping stone for initiation of population based program for preconceptional/prenatal screening of couples.

5 CONCLUSION

With improved genetic diagnostic facilities, there is increasing awareness about prenatal screening and diagnosis for rare genetic disorders. Beta thalassemia screening has become an integral part of good antenatal care. At this juncture when government of India is showing initiative to develop policies for prevention and treatment of genetic disorders, the data obtained from this study will be useful for the medical fraternity and the administrators in initiating steps towards combating this genetic disorder.

ACKNOWLEDGEMENTS

The authors are grateful to the individuals opting in for carrier testing for Spinal Muscular atrophy and also grateful to Mr. Brijesh and Mr. Sandeep for their precious technical support.

    CONFLICT OF INTEREST

    The authors declare no conflict of interest.

    AUTHOR CONTRIBUTIONS

    Dr Shubha R. Phadke conceived the study and planned the methodology and guided Dr Mayank Nilay for collection of data and drafting the manuscript with intellectual input. Dr Mayank Nilay collected the data, compiled the results, tables and drafted the first version of manuscript. Dr Amita Moirangthem participated in the study design, editing the manuscript and intellectual input. Dr Deepti Saxena and Dr Kausik Mandal performed analysis and interpretation of MLPA data with intellectual input. All authors have reviewed critically and approved the final version of the manuscript.

    DATA AVAILABILITY STATEMENT

    The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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