An Iranian familial amyotrophic lateral sclerosis pedigree with p.Val48Phe causing mutation in SOD1: a genetic and clinical report.

OBJECTIVES: Amyotrophic lateral sclerosis (ALS), a fatal progressive neurodegenerative disorder, is the most common motor neuron disease in European populations. Approximately 10% of ALS cases are familial (FALS) and the other patients are considered as sporadic ALS (SALS). Among many ALS causing genes that have been identified, mutations in SOD1 and C9orf72 are the most common genetic causes of the disease. In Iranian patients, it has been shown that SOD1, as compared to C9orf72, plays a much more prominent role. To date, more than 170 mutations have been reported in SOD1. Genotype/phenotype correlation with respect to either different causative genes or different mutations of a specific gene has not been well established.
MATERIALS AND METHODS: Five exons of SOD1 and flanking intronic sequences of an Iranian FALS proband were screened for mutations by direct sequencing. Also, the clinical features of the proband were described.
RESULTS: Heterozygous p.Val48Phe causing mutation was identified in SOD1. Age at onset was 29 years and site of the first presentation was the lower extremity in the proband.
CONCLUSION: The p.Val48Phe causing mutation appears to cause early onset of ALS.

Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by dysfunction and degeneration of both upper motor neurons (UMNs) in the cortex and lower motor neurons (LMNs) in the brainstem and spinal cord that leads to muscular paralysis and ultimately death (1-3). It is the most common motor neuron disease in European countries (4, 5). Its incidence and prevalence in these countries are 1-2/100,000/year and 4-13/100,000/year, respectively (6, 7). Its clinical features depend on several factors, including age at onset of symptoms (one to 94 years) (8, 9), site of onset of symptoms (limbs or bulbar) (10), rate of progression (11, 12), and survival time (few months to over 10 years) (6, 10) . The ultimate cause of death in ALS patients is usually respiratory failure.

Genetics is the source of at least a part of the variability associated with ALS. The majority of patients are sporadic (SALS), while 1-13% of cases in different epidemiological studies were reported to have more than one affected individual in their families and such cases are known as familial ALS (FALS) (13). The mode of inheritance in FALS families is usually autosomal dominant. Mean age at onset of FALS patients is approximately 10 years lower than SALS patients, but they are clinically indistinguishable (9). To date, at least 19 ALS causing genes have been identified (http://alsod.iop.kcl.ac.uk/) (14-16) and approximately, 50-60% of FALS patients carry mutations in these genes (6). Mutations in SOD1 and C9orf72 are the most common causes of disease, although their relative contributions vary in different populations (11, 17-24). SOD1 and C9orf72 are, respectively, the first and one of the most recently identified ALS genes (17, 18, 24). Recently, we showed that mutations in SOD1 are more common than mutations in C9orf72 among Iranian ALS patients (11, 21).

Here, we describe the clinical features of an Iranian FALS patient who harbors a mutation in SOD1 that causes p.Val48Phe. Before, the mutation was once reported in an Italian patient, but detailed clinical features of the patient were not presented (25).

Materials and Methods

This project was performed in accordance with the Helsinki Declaration and approved by the ethics board of the University of Tehran, Tehran, Iran. All participants or their responsible guardians consented to participate after being informed about the project.

Based on El Escorial criteria (26), the proband was diagnosed as definite ALS by a neurologist (SN) in Neuromuscular Clinic of Shariati Hospital, Tehran, Iran. He belonged to a large FALS pedigree that in addition to the proband, includes six ALS patients from four generations (Figure 1). DNA of the proband was isolated according to the standard phenol-chloroform method. Five exons of SOD1 were amplified by polymerase chain reaction (PCR) (Supplementary Table 1, 2) (11). The sequences of all primers that were used are available upon request. All PCR products were sequenced with the same primers that were used in the PCRs, using the ABI big dye chemistry and an ABI Prism 3700 instrument (Applied Biosystems, Foster City, CA). SOD1 reference sequences were NC_000021.8, NM_000454.4, and NP_000445.1. Upon identification of the c.142G>T variation that affects p.Val48Phe in the encoded protein, the variation was screened in 100 Iranian control individuals who were over 60 years old using an allele specific PCR protocol. To assess conservation of p.Val48, amino acid sequences of SOD1 proteins from 17 species were obtained from Uniprot; http://www.uniprot.org/uniprot/ and aligned using ClustalW2 software; http://www.ebi.ac.uk/Tools/msa/clustalw2/.

Figure 1.

ALS164 pedigree. ■ and ● , ALS affected individuals; □ and ○ , asymptomatic individuals; arrow shows proband; present age for some individuals is shown

Additionally, the SIFT; http:// blocks.fhcrc. org/sift/ SIFT.html, PolyPhen; http://genetics.bwh.harvard.edu/pph2, Panther; http://www.pantherdb.org/tools/ csnpScoreForm.jsp and SNAP; https://rostlab.org/ services/snap/submit bioinformatics tools were used to predict the potential pathological effects of p.Val48Phe on the encoded protein.

Results

Genetic analysis

In the encoded SOD1 protein, c.142G>T variation that causes p.Val48Phe was observed in the heterozygous state in the DNA of the proband (Figure 2). No additional variation was detected. The only surviving affected individual in the pedigree (III-1) lives in Europe and was not available for genetic analysis. Furthermore, segregation analysis in the pedigree was not possible because none of the unaffected members of the pedigree consented to genetic analysis; they did not want to know whether or not they carried the mutated allele. However, the c.142G>T variation was not observed in 100 unrelated healthy elderly Iranian control individuals. Furthermore, valine at positions corresponding to p.48 in the human SOD1 protein is well conserved across species from Caenorhabditis elegans to Homo sapiens (Table 1). The SIFT, PolyPhen, Panther and SNAP tools predicted, respectively, that the substitution is damaging, probably damaging, deleterious, non-neutral. Before, the same variation was once reported as the cause of ALS in an Italian ALS family (25). All togethS1er, our data led us to conclude that the p.Val48Phe causing variation in SOD1 was the probable cause of ALS in the proband and his affected relatives. The inheritance pattern of ALS in the pedigree suggests an autosomal dominant mode of inheritance, consistent with observation of a single mutated allele in the proband (Figure 1).

Figure 2.

DNA sequence chromatograms showing the C.142G>T mutation and the wild type sequence. The mutation that causes p.Val48Phe is evident in the in the heterozygous state in the chromatogram of the proband

Table 1

Conservation of p.Val48 in SOD1 proteins

Organism	Seq ID*	Amino acid sequence**
Homo sapiens	P00441	TEGLHGFHVHEFGDNTA
Pan troglodytes	P60052	TEGLHGFHVHEFGDNTA
Macaca mulatta	Q8HXQ0	TEGLHGFHVHQFGDNTQ
Bos taurus	P00442	TEGDHGFHVHQFGDNTQ
Equus caballus	P00443	TKGDHGFHVHEFGDNTQ
Cavia porcellus	P33431	VEGKHGFHVHEFGDNTQ
Sus scrofa	P04178	AEGDHGFHVHQFGDNTQ
Ovis aries	P09670	TEGDHGFHVHQFGDNTQ
Canis familiaris	Q8WNN6	TEGEHGFHVHQFEDXTQ
Oryctolagus cuniculus	P09212	TEGLHEFHVHQFGDNRQ
Rattus norvegicus	P07632	TEGEHGFHVHQYGDNTQ
Mus musculus	P08228	TEGQHGFHVHQYGDNTQ
Gallus gallus	P80566	SDGDHGFHVHEFGDNTN
Lampanyctus crocodilus	P81036	APGLHGFHVHAFGDNTN
Prionace glauca	P11418	TPGKHGFHVHAFGDNTN
Xiphias gladius	P03946	TPGEHGFHVHGFGDNTN
Caenorhabditis elegans	P34697	TPGLHGFHVHQYGDSTN

Clinical data

The ALS patient who was studied here is a member of a FALS pedigree (ALS164) that includes seven ALS-diagnosed patients. The male:female ratio of the patients is 3:4. Five patients had died before the start of the study, and now the proband is also deceased. Available clinical information on five affected members of the pedigree belonging to generations III and IV is presented in Table 2. The average of age at onset of symptoms was 34.6 years (range: 29-45 years). Four patients died 2.5 to 3 years following the onset of symptoms, and the earliest presentations involved the limbs in these individuals. Presentation in the fifth patient (III-1) was bulbar; age at onset for this individual was approximately 13 years more than the average age at onset of the other patients. Patient III-1 is now, two years after the onset, in the final stages of disease. He breathes with the help of a ventilator and is completely paralyzed, unable to speak and swallow. More detailed clinical data on the proband is presented below.

Table 2

Clinical features amyotrophic lateral sclerosis patients of pedigree ALS164

Individual ID	Sex	Age atonset (years)	Present age (years)	Age at death (years)	Disease duration (years)	Site ofonset
I-2	F	?	Dead	?	?	?
II-1	M	?	Dead	?	?	?
III-1	M	45	47	Alive	__	Bulbar
III-5	F	32	Dead	35	3	Lower extremity
III-7	F	35	Dead	37.5	2.5	Lower extremity
IV-11	F	32	Dead	35	3	Upper extremity
IV-13	M	29	Dead	32	3	Upper extremity

F: female; M: male

The proband was a 31-year-old man (IV-13, Figure 1) who presented with a two year history of weakness and atrophy of the limbs which had been started in the left hand and gradually progressed sequentially to involve the right leg, the right hand and the left leg. He mentioned that there was muscle twitching at the beginning, but it was disappeared after a few months. He had no sensory complaint or sphincter dysfunction. Past medical history was unremarkable.

Neurological examination showed normal mental state, tongue atrophy and fasciculation and wasting of left upper extremity. Asymmetric quadriparesis which was more severe in left upper and right lower extremities was seen. Biceps and triceps reflexes were increased on the right side, and were absent on the left side. Right knee and ankle jerks were also absent. Plantar reflexes were downward and sensory examination was intact.

Electromyography that was performed two years after onset of symptoms, showed denervation, fasciculation and reinnervation in various muscles innervated by cranial, cervical, thoracic and lumbar segments. Sensory potentials were normal. The findings were interpreted as definite motor neuron disease according to Awaji criteria (27). Laboratory studies were normal, as were results of brain and cervical spine magnetic resonance imaging (MRI).

Riluzole 50 mg two times a day was started. The patient had progressive deterioration.

He voluntarily entered a clinical trial of autologous mesenchymal stem cell transplantation with intraspinal injection (IRCT201107221696N3). Repeated electro-myography-nerve conduction velocity (EMG-NCV) performed prior to transplantation evidenced reduced compound muscle action potential (CMAP) motor amplitude as compared to his first electromyography. Three months after transplantation, his pulmonary function test showed a forced vital capacity of 75%. The patient remained in a stable and good condition during a four months follow-up. Then, one night he developed severe dyspnea, was admitted to the hospital with a possible diagnosis of pulmonary emboli, and died a few hours later. The details of the transplantation protocol are not presented here.

Discussion

SOD1 encodes copper-zinc superoxide dismutase, which is an evolutionarily highly conserved enzyme that catalyzes the conversion of toxic superoxide anion to hydrogen peroxide and molecular oxygen. In various studies, mutations in SOD1 were observed in 12-23% of FALS patients (average: 20%) and in 0 to 7 percent of SALS patients (average: 3%) (13, 15, 28). In Iranian ALS patients, these mutations were found in 38.5% of the FALS probands, and 4.25% of the SALS cases (11,12). Over 170 different SOD1 mutations have been reported so far (11, 14, 21, 22). While it has been generally difficult to establish clear genotype- phenotype correlations for specific mutations and even for different causative genes, a few exceptions exist. Mutations in SOD1 that cause p.Asp90Ala and p.Leu144Ser are associated with long survival time (11, 29), while p.Ala4Val and p.Gly85Ser are associated with rapid progression of disease (30).

In the present study, we described an Iranian FALS pedigree. The proband of the pedigree harbored a mutation in SOD1 that causes p.Val48Phe in the encoded protein. ALS inheritance in the pedigree was autosomal dominant, without evidence of anticipation. Before, the p.Val48Phe mutation was once reported in an Italian ALS family (25). Although detailed clinical findings were not presented in the earlier finding, age at onset of symptoms in the proband was reported to be about 36 years. This is close to age at onset in patients of ALS164 pedigree (average: 34.6 years). The father of the Italian proband had died from ALS at the age of 39. Therefore, it seems that the p.Val48Phe mutation causes early onset of disease. Age at onset, survival time and limb onset presentation were notably uniform among four of five patients in the ALS164 pedigree. The fifth patient (III-1) differed from the others and had a much higher age at onset and a bulbar presentation. These observations provide evidence that the p.Val48Phe mutation can result in different clinical features even within a single pedigree. The efficacy of the autologous mesenchymal stem cell transplantation in the proband could not be assessed.

Conclusion

As stated above, a clear genotype-phenotype correlation exists for only a few ALS causing SOD1 mutations. Based on clinical data on the Iranian family which was described here and the available data on a previously reported Italian ALS patient who harbored the same mutation, it appears that p.Val48Phe causing mutation in SOD1 mutation causes ALS with an early onset. This having been said, there was some variability in just how early symptoms manifested. Age at onset of four out of five patients ranged between 29 and 36 years while it was notably higher (45 years) for one of them. Limb onset presentation was a common feature among four out of five Iranian patients, but onset was bulbar in the patient who had showed the latest onset. It can be concluded that while the window of clinical presentation for the p.Val48Phe mutation is relatively narrow, particularly with respect to age at onset, it is not strictly uniform.