Letters to the Editor

PCR-based DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous deletion or intragenic mutation of the SMN1 gene. It is well-known that high copy number of its homologous gene, SMN2, modifies the phenotype of SMN1-deleted patients. However, in the patients with intragenic SMN1 mutation, the relationship between phenotype and SMN2 copy number remains unclear.

We have analyzed a total of 515 Japanese patients with SMA-like symptoms (delayed developmental milestones, respiratory failures, muscle weakness etc.) from 1996 to 2019. SMN1 and SMN2 copy numbers were determined by quantitative polymerase chain reaction (PCR) method and/or multiplex ligation-dependent probe amplification (MLPA) method. Intragenic SMN1 mutations were identified through DNA and RNA analysis of the fresh blood samples.

A total of 241 patients were diagnosed as having SMA. The majority of SMA patients showed complete loss of SMN1 (n = 228, 95%), but some patients retained SMN1 and carried an intragenic mutation in the retaining SMN1 (n = 13, 5%). Ten different mutations were identified in these 13 patients, consisting of missense, nonsense, frameshift and splicing defect-causing mutations. The ten mutations were c.275G > C (p.Trp92Ser), c.819_820insT (p.Thr274Tyrfs*32), c.830A > G (p.Tyr277Cys), c.5C > T (p.Ala2Val), c.826 T > C (p.Tyr276His), c.79C > T (p.Gln27*), c.188C > A (p.Ser63*), c.422 T > C (p.Leu141Pro), c.835-2A > G (exon 7 skipping) and c.835-3C > A (exon 7 skipping). It should be noted here that some patients with milder phenotype carried only a single SMN2 copy (n = 3), while other patients with severe phenotype carried 3 SMN2 copies (n = 4).

Intragenic mutations in SMN1 may contribute more significantly to clinical severity than SMN2 copy numbers.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by homozygous deletion of SMN1 exons 7 and 8. However, exon 8 is retained in some cases, where SMN2 exon 7 recombines with SMN1 exon 8, forming a hybrid SMN gene. It remains unknown how the hybrid SMN gene contribute to the SMA phenotype.

We analyzed 515 patients with clinical suspicion for SMA. SMN1 exons 7 and 8 deletion was detected by PCR followed by enzyme digestion. Hybrid SMN genes were further analyzed by nucleotide sequencing. SMN2 copy number was determined by real-time PCR.

SMN1 exon 7 was deleted in 228 out of 515 patients, and SMN1 exon 8 was also deleted in 204 out of the 228 patients. The remaining 24 patients were judged to carry a hybrid SMN gene. In the patients with SMN1 exon 7 deletion, the frequency of the severe phenotype was significantly lower in the patients with hybrid SMN gene than in the patients without hybrid SMN gene. However, as for the distribution of SMN2 exon 7 copy number among the clinical phenotypes, there was no significant difference between both groups of SMA patients with or without hybrid SMN gene.

Hybrid SMN genes are not rare in Japanese SMA patients, and it appears to be associated with a less severe phenotype. The phenotype of patients with hybrid SMN gene was determined by the copy number of SMN2 exon 7, as similarly for the patients without hybrid SMN gene.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by homozygous mutations in the SMN1 gene. SMA has long been known to be the most common genetic cause of infant mortality. However, there have been no reports on the epidemiology of infantile SMA (types 1 and 2) based on genetic testing in Japan. In this study, we estimated the incidence of infantile SMA on Shikoku Island, which is a main island of Japan and consists of four prefectures: Ehime, Kagawa, Tokushima and Kochi.

A questionnaire was sent to 91 hospitals on Shikoku Island to investigate the number of SMA infants born from 2011 to 2015. A second questionnaire was then sent to confirm the diagnoses of SMA based on clinical and genetic features.

Responses were received from all of the hospitals, and four patients were diagnosed with infantile SMA among 147,950 live births. We estimated the incidence of infantile SMA patients as 2.7 per 100,000 live births (95% confidence interval, 0.1–5.4). A comparison of the four prefectures indicated that the incidence of infantile SMA was significantly higher in Ehime Prefecture than in the other three prefectures; 5.6 per 100,000 live births (95% confidence interval, −0.7 to 11.9) in Ehime Prefecture and 1.1 per 100,000 live births (95% confidence interval, −1.0 to 3.1) in the other prefectures.

We estimated the incidence of infantile SMA in an isolated area of Japan. For more precise determination of the incidence of infantile SMA, further studies that include neonatal screening will be needed.

Los avances en el estudio genético de las enfermedades neuromusculares hereditarias (ENM), a través de variadas metodologías de biología molecular, han permitido arribar, en un número creciente de pacientes, a un diagnóstico etiológico de certeza, acortar el tiempo de estudio en cada caso, ofrecer un correcto asesoramiento genético familiar, establecer relaciones genotipo-fenotipo precisas que brinden eventualmente pautas de pronóstico y tratamiento personalizados, agrupando además cohortes de pacientes para su potencial participación en protocolos clínicos específicos y/o para recibir tratamientos farmacológicos personalizados. En este artículo presentamos un resumen de los estudios de biología molecular usados en pacientes afectados de las ENM más frecuentes. El artículo está fundamentalmente dirigido a neurólogos clínicos, ya sean estos pediátricos o de adultos, quienes, frente a un paciente en el que sospechan una ENM, deben solicitar al laboratorio de biología molecular el estudio genético más apropiado, analizar con el paciente el resultado obtenido y complementar la conducta terapéutica basándose en dicho resultado. El estudio genético solicitado debe brindar las mejores posibilidades diagnósticas para el paciente, en el menor tiempo y con el menor costo posible, beneficiando así al paciente y a los sistemas de salud.

Advances of modern molecular biology methodologies for the genetic study of patients with hereditary neuromuscular diseases (NMD), has resulted in a number of benefits for an increasing number of patients: accurate etiological diagnosis, shorter diagnosis times, proper genetic counseling, precise genotype-phenotype relationships that allow for adequate prognosis and treatment options, and the possibility to group cohorts of patients for their potential participation in specific clinical protocols and/or to receive personalized pharmacological treatments. In this article, we present a summary of the molecular biology studies used in patients affected by the most frequent forms of NMD. Our aim is to assist pediatric or adult neurologists to request the most appropriate genetic study when treating a patient with a potential NMD, and to critically analyze the corresponding results to offer appropriate therapeutic alternatives. The requested genetic study must provide the best diagnostic possibilities for the patient, in the shortest time and at the lowest possible cost, thus benefiting the patient and the health systems.