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. 2023 Feb 24;13:3202. doi: 10.1038/s41598-023-30449-7

A comprehensive overview of SMN and NAIP copy numbers in Iranian SMA patients

Shahram Savad 1,✉, Mahmoud Reza Ashrafi 2,3, Niusha Samadaian 1, Morteza Heidari 2,4, Mohammad-Hossein Modarressi 5, Gholamreza Zamani 4, Saloomeh Amidi 1, Sarang Younesi 6, Mohammad Mahdi Taheri Amin 6, Pourandokht Saadati 6, Alireza Ronagh 7, Hossein Shojaaldini Ardakani 8, Solat Eslami 10,9, Soudeh Ghafouri-Fard 11,✉
PMCID: PMC9957985  PMID: 36828874

Abstract

Spinal muscular atrophy (SMA) is among the most common autosomal recessive disorders with different incidence rates in different ethnic groups. In the current study, we have determined SMN1, SMN2 and NAIP copy numbers in an Iranian population using MLPA assay. Cases were recruited from Genome-Nilou Laboratory, Tehran, Iran and Pars-Genome Laboratory, Karaj, Iran during 2012–2022. All enrolled cases had a homozygous deletion of exon 7 of SMN1. Moreover, except for 11 cases, all other cases had a homozygous deletion of exon 8 of SMN1. Out of 186 patients, 177 (95.16%) patients showed the same copy numbers of exons 7 and 8 of SMN2 gene. In addition, 53 patients (28.49%) showed 2 copies, 71 (38.17%) showed 3 copies and 53 patients (28.49%) showed 4 copies of SMN2 gene exons 7 and 8. The remaining 9 patients showed different copy numbers of exons 7 and 8 of SMN2 gene. The proportions of SMA patients with different numbers of normal NAIP were 0 copy in 73 patients (39.24%), 1 copy in 59 patients (31.72%), 2 copies in 53 patients (28.49%) and 4 copies in one patient (0.5%). These values are different from values reported in other populations. Integration of the data of the SMN1/2 and NAIP genes showed 17 genotypes. Patients with genotype 0-0-3-3-1 (0 copies of SMN1 (E7,8), 3 copies of SMN2 (E7,8) and 1 copy of NAIP (E5)) were the most common genotype in this study. Patients with 0-0-2-2-0 genotype were more likely to have type I SMA. The results of the current study have practical significance, particularly in the genetic counseling of at-risk families.

Subject terms: Cell biology, Genetics, Molecular biology

Introduction

Spinal muscular atrophy (SMA) is among the most common autosomal recessive disorders with an incidence rate of about 1 in 6000–10,000 live births. This disorder is described by degeneration of alpha motor neurons in the spinal cord and the medulla oblongata, leading to symmetrical proximal muscular atrophy. Heterozygous healthy carriers for this disorder have a frequency of 1 in 35 in the general population1. Based on the age of onset and reached motor functions, this disorder is classified into four clinical types, namely severe, intermediate, mild and adult-onset types being enumerated as types I to IV, respectively2. From a genetics point of view, this autosomal recessive disorder is caused by the dysfunction of the survival motor neuron (SMN) gene which is located on chromosome 5q13.2. This gene has two versions, namely SMN1 and SMN2. The former produces a full-length transcript. These two versions are different from each other in only five nucleotides. Homozygous deletion of SMN1 exon 7 is responsible for clinical disorder in approximately 94% of cases3. SMN2 has a partial function and can compensate homozygous deletions of SMN1 to some extent4.Therefore, copy numbers of SMN2 affect severity of SMA. Copy number of another gene located on chromosome 5q13.2, namely the neuronal apoptosis inhibitory protein (NAIP) gene has also been shown to be associated with severity of SMA5.

Variations in copy numbers of SMN1 and SMN2 have been reported in SMA patients from different populations. Moreover, different deletions and rearrangements have been detected in different ethnic groups6–8. Thus, identification of SMN1, SMN2 and NAIP copy numbers in SMA patients in each population has a practical significance, particularly in the genetic counseling of at risk families. In the current study, we have determined SMN1, SMN2 and NAIP copy numbers in an Iranian population of SMA patients using MLPA assay.

Methods and patients

Patients

A total of 186 SMA cases were enrolled in this study. Patients were referred to Genome-Nilou Laboratory, Tehran, Iran and Pars-Genome Laboratory, Karaj, Iran during 2012–2022. They were referred to Genome-Nilou laboratory by Iranian SMA Association and neurologists. They came from Tehran and other cities of Iran. All of them were genetically analyzed in Genome-Nilou laboratory. None of the patients used disease modifying therapies. All enrolled cases had a homozygous deletion of exon 7 of SMN1 gene, as confirmed by MLPA assay. Ethical approval for this study has been obtained from the Ethical Committee of Tehran University of Medical Sciences. All methods were carried out in accordance with relevant guidelines and regulations. Informed consent forms were signed by all patients or their parents.

MLPA assay

MLPA was performed using the SALSA MLPA Probemix P021-B1 for detection of deletions or duplication in the exons 7 and 8 of the SMN1, SMN2 and exon 5 of the NAIP genes (MRC-Holland, Amsterdam, Netherlands) as per the manufacturer’s instructions. The resulting fragments were separated using ABI PRISM 3100 (ThermoFisher Scientific, USA) and analyzed by GeneMarker software version 1.959. Peak heights were normalized to control healthy individuals in a similar method to a previous study10, and a deletion or duplication was expected when the normalized peak ratio value was 0 (homozygous deletion), 1 (heterozygous deletion), 3 (heterozygous duplication) and occasionally 4 (heterozygous triplication or homozygous duplication). Each experiment included 4 controls; 2 normal controls, 1 carrier and 1 affected person. All of the controls had been confirmed in an external genetic laboratory.

Statistical analyses

GraphPad Prism version 9.0 (GraphPad Software, La Jolla, CA, USA) (https://www.graphpad.com/guides/prism/latest/statistics/stat_checklist_kw.htm) was used for statistical analysis. Kruskal–Wallis test was performed to detect the relationship between copy number of exons 7 and 8 of SMN2 and deletion in exon 5 of NAIP gene, and SMA subtypes and age at onset. The quantitative data was expressed as mean ± standard deviation. The count data was expressed as the rate and frequency. P value less than 0.05 was considered statistically significant. To compare the distribution of clinical phenotypes (SMA subtypes) between patient groups with/without parental relationship, we used Chi-square (2 × 3 contingency table) (https://www.graphpad.com/quickcalcs/chisquared1.Chi-square/).

Results

General information

Based on the age of disease onset and clinical manifestations, 35, 47, 94 and 10 cases were classified as SMA types I–IV, respectively. A total of 114 cases (61.29%) were born to non-consanguineous parents. Others were born to first cousin (55 cases), first cousin once removed (14 cases) and second cousin (3 cases) parents. The study cohort included 83 females and 103 males.

Gene copy numbers in SMA patients

All enrolled cases had a homozygous deletion of exon 7 of SMN1. Moreover, except for 11 cases, all other cases had a homozygous deletion of exon 8 of SMN1. Out of 186 patients, 177 (95.16%) patients showed the same copy numbers of exons 7 and 8 of SMN2 gene. In addition, 53 patients (28.49%) showed 2 copies, 71 (38.17%) showed 3 copies and 53 patients (28.49%) showed 4 copies of SMN2 gene exons 7 and 8. The remaining 9 patients showed different copy numbers of exons 7 and 8 of SMN2 gene. The proportions of SMA patients with different numbers of normal NAIP were 0 copy in 73 patients (39.24%), 1 copy in 59 patients (31.72%), 2 copies in 53 patients (28.49%) and 4 copies in one patient (0.5%). Table 1 shows detailed characteristics of patients cohort.

Table 1.

Detailed characteristics of patients cohort.

Case Type Sex Age of onset Age of diagnosis Genes copy number Parental relationship
Exon7 SMN1 Exon8 SMN1 Exon7 SMN2 Exon8 SMN2 Exon5 NAIP
1 I Female 0M 3M 0 0 2 2 2 First cousins
2 I Female 1M 3M 0 0 2 2 0 Not related
3 I Female 2M 12M 0 0 2 2 0 First cousins
4 I Female 2M 12M 0 0 2 2 0 First cousin once removed
5 I Female 3M 6M 0 0 2 2 0 Not related
6 I Female 3M 12M 0 0 2 2 0 First cousins
7 I Female 3M 18M 0 0 2 2 0 First cousins
8 I Female 4M 4M 0 0 2 2 0 First cousins
9 I Female 4M 16M 0 0 2 2 0 First cousins
10 I Female 4M 2Y 0 0 2 2 0 Not related
11 I Female 5M 9M 0 0 2 2 0 First cousins
12 I Female 5M 12M 0 0 2 2 0 Not related
13 I Female 6M 6M 0 0 2 2 0 First cousins
14 I Female 6M 8M 0 0 2 2 0 Not related
15 I Male 0M 1M 0 0 2 2 0 First cousins
16 I Male 1M 1M 0 0 2 2 0 First cousin once removed
17 I Male 1M 2M 0 0 2 2 0 Not related
18 I Male 1M 3M 0 0 2 2 2 First cousins
19 I Male 1M 4M 0 0 2 2 0 Not related
20 I Male 1M 6M 0 0 2 2 0 Not related
21 I Male 2M 2M 0 0 2 2 1 First cousins
22 I Male 2M 2M 0 0 2 2 0 Not related
23 I Male 2M 4M 0 0 2 2 0 First cousin once removed
24 I Male 2M 9M 0 0 2 2 0 First cousin once removed
25 I Male 3M 4M 0 0 2 2 0 First cousin once removed
26 I Male 3M 10M 0 0 2 2 0 Not related
27 I Male 3M 10M 0 0 2 2 0 Not related
28 I Male 3M 3Y 0 0 2 2 1 First cousins
29 I Male 4M 4M 0 0 2 2 0 First cousins
30 I Male 4M 6M 0 0 3 3 0 First cousins
31 I Male 4M 9M 0 0 2 2 0 Not related
32 I Male 5M 12M 0 0 2 2 0 First cousins
33 I Male 6M 8M 0 0 3 3 2 First cousins
34 I Male 6M 8M 0 0 4 4 0 First cousins
35 I Male 6M 12M 0 0 2 2 1 First cousins
36 II Female 6M 7Y 0 0 3 3 1 Not related
37 II Female 6M 12Y 0 0 2 2 0 Not related
38 II Female 9M 6Y 0 1 3 2 1 Not related
39 II Female 10M 4Y 0 0 2 2 0 Not related
40 II Female 11M 2Y 0 0 3 3 1 Not related
41 II Female 11M 7Y 0 0 3 3 1 Not related
42 II Female 11M 12Y 0 0 3 3 1 Not related
43 II Female 12M 4Y 0 0 2 2 0 First cousins
44 II Female 12M 5Y 0 0 2 2 0 First cousins
45 II Female 12M 5Y 0 1 3 2 1 Not related
46 II Female 12M 11Y 0 0 2 2 0 Not related
47 II Female 12M 12Y 0 0 3 3 1 Not related
48 II Female 12M 23Y 0 0 2 2 0 Not related
49 II Female 12M 4Y 0 0 2 2 0 First cousins
50 II Female 14M 3Y 0 0 3 3 1 First cousins
51 II Female 14M 9Y 0 0 3 3 1 First cousin once removed
52 II Female 14M 10Y 0 0 3 3 0 Not related
53 II Female 14M 24Y 0 0 3 3 1 Not related
54 II Female 15M 2Y 0 0 3 3 1 Not related
55 II Female 15M 9Y 0 1 4 4 2 Second cousins
56 II Female 15M 11Y 0 0 3 3 1 Not related
57 II Female 15M 17Y 0 0 3 3 0 Not related
58 II Female 15M 32Y 0 0 3 3 1 Not related
59 II Female 17M 18Y 0 0 3 3 1 First cousins
60 II Male 6M 4Y 0 0 3 3 0 Not related
61 II Male 7M 9M 0 0 2 2 0 Not related
62 II Male 7M 7Y 0 0 3 3 0 Not related
63 II Male 7M 12Y 0 0 3 3 0 Not related
64 II Male 7M 18Y 0 0 3 3 0 Not related
65 II Male 8M 3Y 0 0 4 4 1 Not related
66 II Male 8M 15Y 0 0 3 3 0 Not related
67 II Male 9M 12M 0 0 3 3 1 Not related
68 II Male 10M 9Y 0 1 3 3 1 Not related
69 II Male 10M 13Y 0 0 3 3 1 Not related
70 II Male 10M 14Y 0 0 3 3 1 Not related
71 II Male 12M 12M 0 0 3 3 1 Not related
72 II Male 12M 9Y 0 0 3 3 1 Not related
73 II Male 12M 10Y 0 0 2 2 0 Not related
74 II Male 12M 11Y 0 0 4 4 1 Not related
75 II Male 13M 7Y 0 0 3 3 1 Not related
76 II Male 14M 29Y 0 0 4 4 2 Not related
77 II Male 15M 2Y 0 0 3 3 1 Not related
78 II Male 15M 7Y 0 0 3 3 0 Not related
79 II Male 16M 13Y 0 0 3 3 1 Not related
80 II Male 17M 10Y 0 0 3 3 1 Not related
81 II Male 18M 4Y 0 0 3 3 1 Not related
82 II Male 22M 13Y 0 0 2 2 0 Not related
83 III Female 20M 16Y 0 0 4 4 0 Not related
84 III Female 2Y 2Y 0 0 3 3 0 Not related
85 III Female 2Y 7Y 0 0 2 2 1 First cousins
86 III Female 2Y 23Y 0 0 4 4 2 First cousins
87 III Female 2Y 25Y 0 0 4 4 0 Not related
88 III Female 2Y 27Y 0 0 2 2 0 Not related
89 III Female 2Y 31Y 0 0 4 4 2 First cousins
90 III Female 2Y 49Y 0 0 4 4 0 First cousin once removed
91 III Female 3Y 4Y 0 0 4 4 0 Not related
92 III Female 3Y 5Y 0 0 3 3 1 Not related
93 III Female 3Y 9Y 0 0 4 4 1 Not related
94 III Female 3Y 13Y 0 0 3 3 1 Not related
95 III Female 3Y 15Y 0 0 3 3 0 Not related
96 III Female 3Y 17Y 0 0 4 4 0 First cousins
97 III Female 3Y 18Y 0 0 4 4 2 Not related
98 III Female 3Y 19Y 0 0 3 3 1 Not related
99 III Female 3Y 27Y 0 0 3 3 1 Not related
100 III Female 3Y 31Y 0 0 4 4 2 First cousins
101 III Female 4Y 4Y 0 0 3 3 1 Not related
102 III Female 4Y 10Y 0 0 3 3 0 First cousins
103 III Female 4Y 26Y 0 0 4 4 2 First cousins
104 III Female 4Y 27Y 0 0 3 3 1 Not related
105 III Female 4Y 32Y 0 0 3 3 1 Not related
106 III Female 4Y 32Y 0 0 3 3 2 Not related
107 III Female 5Y 6Y 0 0 3 3 1 Not related
108 III Female 5Y 9Y 0 0 4 4 2 First cousins
109 III Female 6Y 23Y 0 0 3 3 1 Not related
110 III Female 7Y 13Y 0 0 3 3 1 Not related
111 III Female 7Y 32Y 0 0 4 4 2 First cousin once removed
112 III Female 7Y 38Y 0 0 3 3 1 Not related
113 III Female 8Y 22Y 0 0 4 4 2 First cousins
114 III Female 8Y 24Y 0 0 3 3 0 Not related
115 III Female 8Y 39Y 0 0 2 2 0 Not related
116 III Female 9Y 16Y 0 0 4 4 2 Not related
117 III Female 9Y 21Y 0 0 4 4 2 Not related
118 III Female 9Y 36Y 0 0 2 2 0 First cousins
119 III Female 9Y 36Y 0 0 4 4 2 First cousins
120 III Female 10Y 23Y 0 0 3 3 1 Not related
121 III Female 11Y 28Y 0 0 3 3 1 Not related
122 III Female 13Y 41Y 0 0 2 2 0 Not related
123 III Female 14Y 34Y 0 0 3 3 1 Not related
124 III Female 16Y 41Y 0 0 3 3 0 First cousin once removed
125 III Female 17Y 34Y 0 0 2 2 0 Not related
126 III Female 21Y 32Y 0 0 3 3 1 Not related
127 III Male 18M 38Y 0 0 3 3 1 First cousin once removed
128 III Male 18M 39Y 0 0 4 4 2 First cousins
129 III Male 19M 7Y 0 0 2 2 0 Not related
130 III Male 2Y 5Y 0 0 3 3 1 Not related
131 III Male 2Y 12Y 0 0 4 4 2 First cousins
132 III Male 2Y 16Y 0 0 3 3 0 Not related
133 III Male 2Y 30Y 0 1 4 3 2 Not related
134 III Male 2Y 30Y 0 0 4 4 2 First cousins
135 III Male 2Y 33Y 0 1 4 3 2 Not related
136 III Male 2Y 37Y 0 0 4 4 2 First cousins
137 III Male 2Y 40Y 0 1 4 3 2 Not related
138 III Male 2Y 43Y 0 1 4 3 2 Not related
139 III Male 3Y 5Y 0 0 3 4 2 Not related
140 III Male 3Y 7Y 0 0 2 2 0 Not related
141 III Male 3Y 8Y 0 0 3 3 0 Not related
142 III Male 3Y 10Y 0 0 3 3 1 Not related
143 III Male 3Y 14Y 0 0 3 3 1 Not related
144 III Male 3Y 14Y 0 0 3 3 1 Not related
145 III Male 3Y 15Y 0 0 3 3 1 Not related
146 III Male 3Y 17Y 0 0 4 4 1 First cousins
147 III Male 3Y 24Y 0 0 4 4 2 First cousins
148 III Male 3Y 26Y 0 0 4 4 2 First cousins
149 III Male 3Y 32Y 0 1 3 2 2 Not related
150 III Male 3Y 41Y 0 0 4 4 2 Not related
151 III Male 3Y 43Y 0 0 4 4 2 First cousin once removed
152 III Male 4Y 4Y 0 0 3 3 1 Not related
153 III Male 4Y 10Y 0 0 4 4 2 Not related
154 III Male 4Y 29Y 0 0 4 4 2 Not related
155 III Male 5Y 7Y 0 0 4 4 2 First cousins
156 III Male 5Y 15Y 0 0 2 2 0 First cousins
157 III Male 6Y 20Y 0 0 3 3 2 Second cousins
158 III Male 6Y 30Y 0 0 2 2 0 Not related
159 III Male 7Y 29Y 0 0 3 3 0 Not related
160 III Male 7Y 31Y 0 0 3 3 1 Not related
161 III Male 7Y 31Y 0 0 4 4 2 First cousins
162 III Male 11Y 12Y 0 0 3 3 2 Not related
163 III Male 11Y 33Y 0 0 4 4 2 First cousins
164 III Male 12Y 22Y 0 1 4 3 2 Not related
165 III Male 12Y 35Y 0 0 4 4 2 First cousins
166 III Male 12Y 40Y 0 0 3 3 0 Not related
167 III Male 12Y 50Y 0 0 4 4 2 Not related
168 III Male 13Y 16Y 0 0 3 3 2 First cousins
169 III Male 13Y 23Y 0 0 4 4 2 First cousins
170 III Male 13Y 26Y 0 0 4 4 0 Not related
171 III Male 15y 27Y 0 0 4 4 2 First cousins
172 III Male 15Y 30Y 0 0 4 4 0 First cousin once removed
173 III Male 15Y 43Y 0 0 4 4 0 First cousin once removed
174 III Male 17Y 32Y 0 0 4 4 2 First cousins
175 III Male 18Y 33Y 0 0 4 4 2 Not related
176 III Male 22Y 29Y 0 0 4 4 2 First cousin once removed
177 IV Female 30Y 41Y 0 0 3 3 1 Not related
178 IV Male 19Y 29Y 0 0 4 4 4 First cousins
179 IV Male 20Y 33Y 0 0 4 4 2 First cousins
180 IV Male 20Y 40Y 0 0 2 2 1 Not related
181 IV Male 25Y 45Y 0 0 4 4 2 Second cousins
182 IV Male 25Y 49Y 0 0 4 4 2 Not related
183 IV Male 27Y 42Y 0 2 4 4 2 Not related
184 IV Male 28Y 40Y 0 0 3 3 1 First cousins
185 IV Male 28Y 40Y 0 0 4 4 2 First cousins
186 IV Male 28Y 40Y 0 0 4 4 2 First cousins
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Distribution of SMA patients in different groups of SMA is shown in Table 2. Type III SMA accounts for 50.53% of total cases.

Table 2.

Results of genetic diagnosis of SMA patients.

Clinical type Type I Type II Type III Type IV Total
Number 35 47 94 10 186
Proportion 18.81% 25.26% 50.53% 5.37% 100%
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While exon 7 was absent in all SMA patients of all classes, exon 8 was present in 4 Type II, 6 Type III and 1 type IV SMA cases. In fact, in 94.08% (175/186) of the patients, homozygous deletion of both exons 7 and 8 of the SMN1 gene was reported. Among these, 18.81% (35/186) of patients were diagnosed with SMA Type I, 25.26% (47/186) with Type II, 50.53% (94/186) with Type III, and 5.37% (10/186) with Type IV. In 5.9% (11/186) of the patients, homozygous deletion of the 7th exon and heterozygous deletion of 8th exon of the SMN1 gene were detected (Table 3). There was no correlation between different SMA types and deletion types of exons 7 and 8 of SMN1 gene (P value = 0.31).

Table 3.

SMN1 Exons copy numbers in patients with different clinical types of SMA.

Clinical types Type I Type II Type III Type IV Total
Copy number
EXON 7 0 35 (18.81%) 47 (25.26%) 94 (50.53%) 10 (5.37%) 186 (100%)
1 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)
Total 35 (18.81%) 47 (25.26%) 94 (50.53%) 10 (5.37%) 186 (100%)
EXON 8 0 35 (18.81%) 43 (23.11%) 88 (47.31%) 9 (4.83%) 175 (94.08%)
1 0 (0%) 4 (2.15%) 6 (3.22%) 0 (0%) 10 (5.37%)
2 0 (0%) 0 (0%) 0 (0%) 1 (0.53%) 1 (0.53%)
Total 35 (18.81%) 47 (25.269%) 94 (50.53%) 10 (5.37%) 186 (100%)
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Totally, 27.95% (52/186), 38.7% (72/186), and 28.49% (53/186) of patients had 2, 3, and 4 copies of exons 7 and 8 of the SMN2 gene, respectively (Fig. 1). However, 9 patients showed different normal copy numbers of exons 7 and 8 of SMN2 gene. Three out of nine patients showed 3 copies of exon 7 and 2 copies of exon 8, five out of nine patients showed 4 copies of exon 7 and 3 copies of exon 8 and one patient showed 3 copies of exon 7 and 4 copies of exon 8 of SMN2 gene. In addition, 39.24% (73/186), 32.73% (59/186), 31.72% (53/186) and 0.53% (1/186) of patients had 0, 1, 2 and 4 copies of the exon 5 of the NAIP gene, respectively. The presence of two copies of SMN2 gene was most common in type I patients, accounting for 91.42% (32/35) of these patients. The presence of 3 copies of SMN2 was most common in type II patients, accounting for 72.34% (34/47) of patients. Finally, having 4 copies of this gene was most common in type III and type IV patients, accounting for 48.93% (46/94) and 70% (7/10) of patients, respectively.

Figure 1.

Figure 1

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The percentage of individuals with various numbers of exon 7 of the SMN2 gene.

Figure 1 shows the percentage of individuals with various number of SMN2 gene copies.

Figure 2 shows the percentage of individuals with various numbers of NAIP gene copies. There was a significant difference in the distribution of NAIP gene copy numbers among different types of SMA (× 2 = 69, P < 0.0001). All patients carrying deletion of two copies of NAIP gene had severe (type I) SMA, accounting for 82.85% (29/35) of patients. Having one copy of this gene was most common in type II patients, accounting for 57.44% (27/47) of patients. The presence of two copies of NAIP gene was most common in type III and type IV patients, accounting for 44.68% (42/94) and 60% (6/10), respectively (Fig. 2).

Figure 2.

Figure 2

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The percentage of individuals with various numbers of NAIP gene.

The average age of onset for patients with 2 copies of SMN2 gene (23.86 ± 50.14 month) was significantly lower than that of patients with 3 (50.75 ± 69 month) or 4 (99.25 ± 96.56 month) copies of SMN2 (P < 0.0001) (Fig. 3a).

Figure 3.

Figure 3

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Relationship between copy numbers of exon 7 of SMN2 (a) and NAIP (b) genes and age at onset of patients. A non-parametric Kruskal–Wallis test was used to identify significant association between the age at onset of patients and SMN2 and NAIP genes copy number (* P value < 0.05, *** P value < 0.001 and **** P value < 0.0001).

The average age of onset of SMA in patients with 0 copy of the NAIP gene (33.1 ± 51.98) was also less than that of patients with 1 (50.15 ± 75.81) and 2 (99 ± 96.65) copies (P < 0.0001) (Fig. 3b).

There was a significant difference in the distribution of NAIP gene copy numbers among different types of SMA. All patients carrying deletion of two copies of SMN2 and NAIP genes had severe (type I) SMA.

Chi-square (2 × 3 contingency table) was performed to compare the distribution of NAIP E5 copy numbers between patient’s groups with/without parental relationship (not related vs. related groups). The analysis showed that there was significant difference (× 2 = 25.36, P < 0.0001) in the distribution of NAIP E5 copy numbers in patient’s groups regarding the parental relationship. In fact, 95 out of 115 (82.6%) of patients with no parental relationship had no or one NAIP E5 copy number and 20 (17.4%) of patients with no parental relationship had two NAIP E5 copy numbers. However, among the 71 patients with parental relationships, 27 patients (38%) had no NAIP E5 copy number, 34 patients (47.9%) had two NAIP E5 copy numbers and 10 patients (14.1%) had one NAIP E5 copy number.

There was also a strong significant correlation between copy numbers of SMN2 and NAIP genes (R = 0.68, P < 0.0001) and the copy numbers of SMN2 and NAIP genes had synergistic effect on SMA phenotype.

Integration of the data of the SMN1/2 and NAIP genes showed 17 genotypes. Patients with genotype 0-0-3-3-1 (0 copies of SMN1 (E7,8), 3 copies of SMN2 (E7,8) and 1 copy of NAIP (E5)) were the most common genotype in this study (Table 4). Patients with 0-0-2-2-0 genotype were more likely to have type I SMA.

Table 4.

Relationship between SMN1 (E7and E8) -SMN2 (E7and E8) -NAIP (E5) genotype and clinical phenotype of SMA.

Genotype Case numbers (%) Age of onset, month (mean ± SD) Clinical classification (%) Sex
Type I Type II Type III Type IV
0-0-3-3-1 47 (25.26%) 54.48 ± 78.54 0 (0%) 22 (46.8%) 23 (48.9%) 2 (4.25%)

28 Female

19 Male
0-0-2-2-0 45 (24.19%) 21.42 ± 42.32 27 (60%) 9 (20%) 9 (20%) 0 (0%)

24 Female

21 Male
0-0-4-4-2 37 (19.89%) 113 ± 99 0 (0%) 1 (2.7%) 31 (83.78%) 5 (13.5%)

11 Female

26 Male
0-0-3-3-0 19 (10.21%) 41 ± 52.1 1 (5.26%) 9 (47.36%) 9 (47.36%) 0 (0%)

8 Female

11 Male
0-0-4-4-0 9 (4.83%) 74.75 ± 77.33 1(11.11%) 0 (0%) 8 (88.88) 0 (0%)

5 Female

4 Male
0-0-2-2-1 5 (2.68%) 55 ± 103.8 3 (60%) 0 (0%) 1(20%) 1(20%)

1 Female

4 Male
0-0-3-3-2 4 (2.15%) 82.5 ± 61.19 1 (25%) 0 (0%) 4 (75%) 0 (0%)

1 Female

4 Male
0-0-4-4-1 4 (2.15%) 23 ± 15.09 0 (0%) 2 (50%) 2 (50%) 0 (0%)

1 Female

3 Male
0-1-3-2-1 2 (1.07%) 10.5 ± 2.12 0 (0%) 2 (100%) 0 (0%) 0 (0%)

2 Female

0 Male
0-0-2-2-2 2 (1.07%) 1 ± 0 2 (100%) 0 (0%) 0 (0%) 0 (0%)

1 Female

1 Male
0-1-3-2-2 1 (0.53%) 36 ± 0 0 (0%) 1 (100%) 0 (0%) 0 (0%)

0 Female

1 Male
0-1-4-3-2 5 (2.68%) 48 ± 53 0 (0%) 0 (100%) 5 (100%) 0 (0%)

0 Female

5 Male
0-0-4-4-4 1(0.53%) 228 ± 0 0 (0%) 0 (0%) 0 (0%) 1 (100%)

0 Female

1 Male
0-1-4-4-2 1 (0.53%) 15 ± 0 0 (0%) 1 (0%) 0 (0%) 0 (0%)

1 Female

0 Male
0-2-4-4-2 1 (0.53%) 324 ± 0 0 (0%) 0 (0%) 0 (0%) 1 (0%)

0 Female

1 Male
0-1-3-3-1 1 (0.53%) 10 ± 0 0 (0%) 1 (0%) 0 (0%) 0 (0%)

0 Female

1 Male
0-0-3-4-2 1 (0.53%) 36 ± 0 0 (0%) 0 (0%) 1 (0%) 0 (0%)

0 Female

1 Male
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To compare the distribution of clinical phenotypes (SMA subtypes) between patient’s groups with/without parental relationship, patients’ group was divided into three subtypes I, II and III &IV. Chi square test in the 2 × 3 contingency table analysis provided evidence that there was significant difference (× 2 = 26.12, P < 0.0001) in the distribution of clinical phenotypes (SMA subtypes) in patient’s groups regarding the parental relationship. The frequency of type I patients was higher in patients with parental relationship (first cousins or first cousin once removed) while the frequency of patients with types II and III subtypes was higher in patients with non-consanguineous families (Fig. 4).

Figure 4.

Figure 4

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The distribution of clinical phenotypes (SMA subtypes) between patient’s groups with/without parental relationship. Chi-square (2 × 3 contingency table) was performed to compare the distribution of clinical phenotypes between patient’s groups (not related and related groups).

Discussion

In the current study, we assessed SMN1, SMN2 and NAIP copy numbers in a large population of Iranian patients with SMA. The majority of enrolled patients were born in non-consanguineous families which is consistent with high rate of normal carriers in Iranian population. A previous study in Iranian population estimated a carrier frequency of 5% in this population11. Consistent with this report, a more recent literature review has suggested higher frequency of heterozygous carriers of the SMN1 mutations among Caucasian and Asian populations compared to the Black population12.

In our cohort of patients, all patients except for 11 cases had a homozygous deletion of exon 8 of SMN1. This finding is comparable with the findings in Chinese population2 and some other populations13.

The proportions of SMA cases with different numbers of normal SMN2 copies were 2 copies in 53 (28.49%), 3 copies in 71 (38.17%) and 4 copies in 53 (28.49%). These values are significantly different from those reported by Fang et al. in Chinese population2. They reported the presence of 1–4 normal SMN2 copies in 2 patients (4.8%), 14 (33.3%), 24 (57.1%) and 2 (4.8%) patients in their cohort, respectively2. Amara et al. have reported that 31.3% of Tunisian type I SMA patients carry one copy of SMN2, though all patients of other forms had a minimum of 2 copies14.

The proportions of SMA patients with different numbers of normal NAIP were 0 copy in 73 patients (39.24%), one copy in 59 patients (31.72%), 2 copies in 53 patients (28.49%) and 4 copies in one patient (0.5%). These figures are also different from Fang et al. report in Chinese population as authors reported 0–2 copies in 4 (9.5%), 26 (61.9%) and 12 patients (28.6%), respectively2. Moreover, NAIP has been reported to be absent in the majority of Tunisian SMA type 1 patients14.

Thus, there is significant difference in the copy number of mentioned genes among SMA patients of different populations. This difference might be due to the presence of some founder mutations in each population.

We also compared the copy numbers of SMN2 and NAIP between four classes of SMA patients. These disease-modifying genes have been shown to influence age of onset of SMA patients. These two genes have been shown to be the most important modifier genes whose copy numbers can influence clinical course of SMA. Hassan et al. have shown that the combination of these genes has better performance in prediction of patients' prognosis than using CNVs of exon 7 of SMN2 gene only. While CNVs of exon 7 of SMN2 gene could predict response of patients to genetic therapy, deletion of exon 5 of NAIP gene alone could not predict severity of SMA15. Another study has shown that NAIP deletion is significantly related to the clinical severity of SMA and is a marker for prediction of SMA prognosis16. This finding has also been confirmed in our study, since all patients carrying deletion of two copies of NAIP gene had severe (type I) SMA.

Zhang et al.17 have determined five combined SMN1-SMN2-NAIP genotypes in their cohort of SMA patients with 0-3-1 genotype being the commonest one. Similarly, in our cohort of patients, 0-3-1 genotype had the highest frequency accounting for 26.19% of cases. Moreover, Zhang et al., have reported the synergistic effect of copy numbers of SMN2 and NAIP genes on clinical course of SMA. They have demonstrated association between the combined SMN1-SMN2-NAIP genotypes with fewer copies and earlier disease onset and higher mortality in SMA patients17. Another study in Vietnamese population has shown association between copy numbers of SMN2 and clinical severity of SMA. However, heterozygous NAIP deletion has been found commonly in SMA patients of this population in an independent manner from the clinical phenotype18. The latter finding is not consistent with our study, since we found association between copy numbers of both SMN2 and NAIP genes and age of disease onset in Iranian population. Similar finding has been reported among Malaysian SMA patient19.

Taken together, the current study is the largest and the most comprehensive genetic analysis of Iranian patients that analyzed SMN1, SMN2 and NAIP copy numbers simultaneously. This study also shows the spectrum of SMN2 and NAIP copy numbers in Iranian SMA patients.

Acknowledgements

We would like to thank Iranian SMA Association and Iranian SMA Registry of TUMS for their kind supports in the current study.

Author contributions

S.G.F. and S.S. wrote the draft and revised it. MHH designed and supervised the study. M.R.A., A.R., H.S.A., G.Z., M.H. and S.A. performed the experiment. S.E. and S.Y. analysed the data. M.M.T.A. and P.S. collected the data. All the authors read and approved the submitted version.

Data availability

The datasets generated and/or analysed during the current study are available in the Clinvar repository (https://www.ncbi.nlm.nih.gov/clinvar/?gr=0&term=smn).

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Shahram Savad, Email: Shahram.savad@yahoo.com.

Soudeh Ghafouri-Fard, Email: s.ghafourifard@sbmu.ac.ir.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated and/or analysed during the current study are available in the Clinvar repository (https://www.ncbi.nlm.nih.gov/clinvar/?gr=0&term=smn).

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