The identification of the genotype constitution (SMN1: SMN2 copy numbers) in patients diagnosed with SMA has important implications for the genetic counseling of families with or without a history of the disease. Approximately 94–95% of individuals with SMA are homozygous carriers of a SMN1 exon 7 deletion (OMIM #253300; ORPHA #70). On the other hand, SMN2 is a SMA modifier gene, and a strong inverse correlation between its copy number and disease severity has been demonstrated: with a larger number of copies, the better the prognosis and the later the age of onset [3, 13, 14].
In our study, the number of SMN2 copies and the carrier frequency of the SMN1 deletion in a random sample of the Venezuelan populations were assessed, as well as in families with a presumptive diagnosis of SMA.
Gene dosages
The frequency of carriers of 2 SMN1 copies in the Venezuelan control sample was 87.8%, very similar to the observed pan-ethnic frequencies (84.84%) [4].
It has been reported that about 2–5% of SMA carriers have two SMN1 copies in cis in one chromosome and none in the other (silent carriers 2/0).This could produce a lower detection rate [15, 16]. Neither MLPA nor qPCR techniques allow detection of the carriers of this condition, giving a false negative result. Thus, in partners of known SMA carriers, it is important to test their parents and additional family members to exclude the presence of a cryptic 2/0 genotype [17].
One of the 49 studied control subjects had 1 copy of the gene, being thus a carrier of the deletion. This corresponds to an allelic frequency of 0.01 in this sample (n = 98 chromosomes), for a carrier frequency of 2.04% (1/49) in the Venezuelan populations, being close to those values reported in diverse populations: Brazil 2.7% [18]; USA Caucasoids 2.7%; African Americans 1.1%; Ashkenazi Jews 2.2%; and 1.8% in Asians [19]. In sub-Saharan populations, the frequencies are lower: 1.6% in Nigerians, 0.83% in Kenyans, 0.48% in Malian [20]; in France, it is 2.9% in the general population [21]. Moreover, the frequency of homozygotes with deletion of SMN1 in the Venezuelan sample calculated by extrapolation of the Hardy-Weinberg equation is 1/9091, being similar to several studies, in which the estimated prevalence has been around 1/6000 to 1/10000 livebirths [22, 23].
Around 4% of the control individuals carried 3 copies of SMN1 as has been found in most populations with variable frequencies (5.3 to 41.3%) [24]. Three individuals had 4 or more copies of SMN1; they did not carry any copy of SMN2. This inverse correlation, caused by gene conversion events between both genes [2], suggests that the increase in the number of copies of SMN1 is associated with the number of copies of SMN2 in the general population. Furthermore, 16% of control individuals of the Venezuelan population sample lack SMN2 copies. According to the results obtained in other assessed populations [24], the average of SMN2 = 0 in the main ethnic groups are European = 6.8%, Asian = 3.9%, African = 23.1%, and American = 7.2% (the last category only includes Colombian and Peruvian subjects, total n = 100). The admixed populations of Venezuela have ancestral Amerindian, African, and Spanish genetic contributors, in varying proportions depending on the geographic regions. Our study is the first that assessed SMN copy number variation in a Venezuelan control sample. Although the sample is small (n = 98 chromosomes), it informs about the frequencies in our random admixed populations, which in the studied cohort seems to be close to the American, but higher, and African ones. A larger population sample should thus be studied to confirm these findings.
Genotype-phenotype correlation
SMA is a disease with a wide inter- and intra-family phenotypic heterogeneity. The motor dysfunction associated with the loss of motor neurons and skeletal muscle atrophy is a defining feature of SMA throughout the clinical spectrum of the disease. The symptoms of the studied cases were characterized by generalized muscular hypotonia, areflexia, weakness of the intercostal muscles, progressive proximal weakness, inability to remain seated or standing, scoliosis, and electromyography (EMG) results compatible with SMA. In 10 out of 14 cases, SMA was confirmed by homozygous deletion of SMN1. In a single instance, the index case did not have any SMN1 deletion despite having all the typical features of the disease; he probably is a carrier of a point mutation in the gene, a possibility which should be confirmed by a Sanger sequencing.
The analysis of the number of SMN2 copies in the studied individuals homozygous for the SMN1 deletion showed that, although the phenotype-genotype correlation is quite clear, it is not the only genetic factor influencing it. The correlation coefficient estimated from the SMN2 number of copies and the SMA subtype was 0.416, indicating a significance, but not complete correlation between the genotype and the clinical phenotype, attributable to the eventual presence of positive modifiers, such as the SMN2 variants c.859G>C (p.Gly287Arg) in exon 7 and g.69372304A > G in intron 6; both variants produce a moderate increase in exon 7 inclusion [25, 26]. Additionally, the PLP3 gene is known to influence the phenotype [27]. However, the number of SMN2 copies is the strongest known modifier of the SMA phenotype [3] and it is relevant for current clinical trials using nusinersen (SpinrazTM). This is the first approved drug for SMA treatment. Nusinersen is an antisense oligonucleotide that binds specifically a silencer sequence in intron 7 allowing the inclusion of exon 7 in the mature mRNA from SMN2 [17], increasing thus the amount of a functional full-length SMN protein. The available data show that the efficacy of the treatment is enhanced when it is applied before or soon after the onset of symptoms [28, 29]. Targeted treatment is recommended for all individuals who have two or three copies of SMN2; contrarily, for individuals with four or more copies, targeted treatment can be deferred until symptom onset [30].
A clear SMN2 genotype-phenotype correlation was observed in family 7 (Table 3), which had four affected siblings (Fig. 1), with ages of onset between 6 and 11 months of age. Patient III-6 was born without motor activity and died at 4 months of age with respiratory insufficiency. It was surprising to find that the mothers (II-3 and II-5, Fig. 1) of the affected children despite being also homozygous carriers of the deletion had very few clinical manifestations of the disease: fine tongue tremor and decreased muscle strength in the hands. Quantification of SMN2 copy number revealed 4–5 in each case, clearly illustrating the phenotypic modulation of SMN2 on the disease severity.
On the other hand, the haplotype analysis in the family revealed that a double recombinant event had occurred in the transmission of the affected gene from one of the mothers (II-5, Fig. 1) to two of her affected children (III-7 and III-8). The presence of a large inverted repeat as well as multiple smaller repeats in the 5q13 region makes the SMN locus highly susceptible to unequal recombination, causing de novo deletions of SMN1 [2]. This mechanism could be acting in this family, which showed an unexpected number of independent deletion carriers. Husbands of the affected mothers (II-3 and II-5, Fig. 1) are not biologically related, carrying different in-phase haplotypes.
The findings in this family show the importance of performing genetic tests to different relatives at risk in specific families, even if they did not have clinical manifestations of the disease. Unfortunately, grandfather I-1 could not be studied but his spouse I-2 was informative enough for inferring genotype origins in their immediate descendants.
Geographic distribution
There was neither close nor remote kinship between the parents in any of the families. Regarding the possible geographic aggregation of SMN1 gene deletion, in only one family, it was observed an eventual “geographic focus” [31], since two grandparents from different ancestral lines of the index case were born in Maracay, State of Aragua. Ancestors of the control individual, carrier of the SMN1 deletion, were born in the central states of the country: Guárico (Calabozo) and Aragua (Villa de Cura), this latter city being very close to Maracay city, suggesting a higher mutation prevalence in that eventual geographic focus. The remaining families had quite diverse places of birth of their ancestors throughout the country, without any clear or even suspected aggregation.