Duchenne Muscular Dystrophy in Kazakhstan: A Journey from Diagnosis to the Treatment, the Biases and Achievements

1INTRODUCTION

Duchenne Muscular Dystrophy (DMD) is a rare neuromuscular X-linked disorder that belongs to a group of disorders known as dystrophinopathies. DMD is caused by pathogenic variations in the dystrophin gene, which is one of the largest known human genes. DMD gene variations lead to an absence or reduction of the dystrophin protein. Dystrophin is notably crucial for the stability of the membrane in skeletal muscle. Dystrophin isoforms are also expressed in the brain which accounts for cognitive impairment often seen in DMD. As such, the lack of functional dystrophin impairs the structure and function of myofibres, something critical for the physiological growth and maintenance of muscle tissue [1].

To date, more than 3,000 various disease-causing variations of the DMD have been reported. The majority of them are deletions that account for 65–78%of these variations. In most deletions, mutations may lead to alteration of the open reading frame (ORF) leading to a complete absence of dystrophin. The rest of the variations consists of duplications (6%–10%), point mutations (missense, nonsense, and splice site variations) (10–25%), or other smaller rearrangements: insertions/deletions, small inversion. Approximately 2%of mutations are caused by complex mutations and deep intronic rearrangements [3].

Since 2014 several disease-modifying medications that affect disease progression have been approved. For example, Ataluren was authorised by the European Medicine Agency in 2014 [4, 5], Eteplirsen was approved by the U.S. Food and Drug Administration in 2016, and more recently golodirsen, viltolarsen and casimersen [6]. Such medications except viltolarsen are all in the list of clinical guides for treatment of approved for DMD/BMD in Kazakhstan.

Kazakhstani doctors have started to pay closer attention to DMD diagnosis over the last 5–7 years. As this diagnosis is not very common, based on the worldwide prevalence of disease (1 : 3500 alive born boys a year by National Organization for Rare disorders, 2019) we expect that there should be around 580 children in Kazakhstan with such a diagnosis.

This retrospective study aims to describe the establishment of diagnostic approaches based on the analysis of some clinical issues such as onset, stage of disease, CK level and the spectrum of dystrophin gene variations in DMD patients in Kazakhstan.

2MATERIALS AND METHODS

In this retrospective study conducted in Kazakhstan, data has been obtained from the Multidisciplinary reference centre on neuro-muscular disorders located in Astana, Kazakhstan. 84 patients from (age 2–19 years) have been included in the study. All patients were males. Although the sample studied is not fully representative of the whole Kazakh population, it still offers a valuable snapshot of the recorded cases of DMD diagnosis and treatment in Kazakhstan. The enrolment criteria for the study was a DMD diagnosis, identified genetic mutation by Multiple Ligation Probe Amplification and/or by sequencing of DMD gene or NGS analysis. Written consent was provided by the parents, when patients proceeded with the genetic analysis. The MLPA analysis was conducted in Kazakhstan (MRC-Holland Salsa MLPA probemix P034/P035) and in Germany (Centogene variant classification (based on ACMG recommendations)). The statistical data has been processed by the Excel program.

3RESULTS

84 patient’s records were analysed. The age of patients varied between 2 years and 19 years. The median age of patients was 10 years at registration.

The onset of disease has been estimated in 58 cases (motor signs, milestones delay and transaminase elevation). The average age of onset of disease was 3 years and 4 months. The minimal age of onset was 4 months (liver transaminase elevation) and the maximal age of disease onset was 96 months (8 years). 23 records do not contain data about the time when the first clinical manifestations were noted. Three cases among 84 were diagnosed at a presymptomatic stage within the framework of the family screening.

The medical records contain data on clinical stages in almost all cases except one. On physical examination, the stage when the patient’s diagnosis was established as ambulatory –60, non-ambulatory –20 and 1 patient was on the stage of diagnosis estimation (4 months). 2 patients were discussed on Board commission twice and the clinical stage was initially ambulatory, which deteriorated to non-ambulatory later on (Table 1).

The average age when the genetic diagnosis has been firmly established was 7 years and 6 months. The minimal age of genetic confirmation was 4,5 months and the maximal age when the genetic test was performed was 17 years.

There were 49 cases (58,33%) of gross rearrangements, of which 47 (55,95%) were deletions (one exon or multiple exons) and 2 (2,38%) were duplications. Nonsense mutations were identified in 25 cases (29,7%). Combined mutation was diagnosed in 1 case (1,19%). There were 4 frameshift mutations (4,76%), as well. 2 mutations (2,38%) among all pathogenic variations were substitution and splicing. 1 case (1,19%) of intronic mutation was recorded (Tables 2, 3, Diagram 1)

Table 3

Spectrum and frequency of deletion cases

Diagram 1

The distribution of DMD pathogenic variations among patients (total number is 84 (in %)).

The level of creatine phosphokinase in primary years of disease was known in 61 patients. The average level of creatine phosphokinase is –8 516,66 IU.

4DISCUSSION

The diagnostic method in Kazakhstan remains the same as in the other countries. Initially, since 1995, the PCR analysis of 19 exons has been launched, then since 2011 the diagnostic method has been modernised and MLPA analysis has been implemented. A biochemical blood analysis (creatine phosphokinase (CK), lactate dehydrogenase (LDH), liver transaminase levels as ALT and AST), an electromyography and MLPA are conducted on patients who exhibit clinical manifestations, have specific complaints, and/or have a positive family history. Such diagnostic procedures are all available in Kazakhstani laboratories.

It should be noted, however, that while there is recognition that an electromyography is limited in the diagnosis of DMD and is not widely used internationally, its practice in Kazakhstan, as a way to narrow down the search for a diagnosis, has remained active since the Soviet era. The negative MLPA analysis in clinically manifested children gives the opportunity to proceed with NGS in order to reveal the pathological variations. The NGS method is conducted in the Centogene laboratory (Germany). Ideally, the patient with a positive genetic test should then be directed to a paediatric neurologist, who specialises in neuromuscular diseases. In reality, even with confirmed clear diagnostic algorithms many medical professionals are not familiar with neuromuscular conditions, naturally leading to wrong diagnoses and inappropriate management.

In Kazakhstan, management of patients with any neuro-muscular disorder is a burning issue now due to an increasing number of patients with muscle diseases, although the alertness of doctors is virtually lacking regarding this rare condition. Many of these patients have wrong diagnosis and they are treated more aggressively than needed due to heterogenicity of muscle diseases. In Kazakhstan, international guidelines for diagnostic and treatment approaches to Duchenne muscle dystrophy were just recently accepted, and we started to give patients more or less proper care including steroids, respiratory and nutritional support. Nevertheless, other types of muscle diseases are still underdiagnosed or even undiagnosed due to lack of knowledge and absence of a system for neuromuscular service in Kazakhstan. Proper diagnosis and management for such kinds of rare diseases are mostly dependent on the growth of scientific knowledge and the development of new therapies. Importantly, the status of some of those diseases are now moved from the untreatable to treatable side. Needless to say, it is very important to establish a system for muscle diseases in order to provide proper health care to patients through scientifically-proven approaches, such as imaging, immunohistochemistry, and molecular studies.

As a result of a lack of DMD specialists in Kazakhstan but the necessity in solving the issue at the same time, the national expert-consulting board at Multidisciplinary reference centre on neuro-muscular disorders was created in Astana, Kazakhstan in August 2020. The national expert-consulting board consists of 10 specialists, whose work focuses on neuro-muscular disorders. This Board considers the clinical, functional and genetic status of the patient, advises a diagnostic method, prescribes therapy and manages treatment.

Our study shows that the mean age of patients with DMD, when the doctors started to discuss a target treatment possibility, is 10 years. The average age for the onset of the DMD in Kazakhstani patients, according to clinical observations, is 3 years and 3 months. But the decision to implement target treatment is only possible when the doctors have a genetic confirmation of mutation at DMD gene. Our data shows that the genetic diagnosis in Kazakhstan is performed later, demonstrating a delay of diagnosis and treatment for more than 4 years. Since the discovery of the DMD gene in 1987, significant progress has been made in genetic testing using DNA analysis for DMD, so that it is now possible to identify DMD mutations from blood specimens in over 95%of patients with DMD [8]. Several clinic-based studies spanning more than a decade show a mean age for diagnosis of DMD between 4 and 5 years [9–12]. The delay of diagnosis in Kazakhstan results in lost opportunities for timely genetic counselling and initiation of steroids and target treatment. Some patients and doctors are afraid of steroids’ side effects, which is a treatment stigma. According to reports of the national expert-consulting board at Multidisciplinary reference centre on neuro-muscular disorders 4 patients among 24 non-ambulant have been prescribed Ataluren. Our data shows that the patients start to become non ambulant at the age of 9–10 years. Most of the patients can keep ambulation up to 13–14 years of age and we have not seen ambulant patients older than 14–15 years. Such findings reflect known information about the natural duration of the disease. According to the publications, the disease progresses very quickly and usually the patients are in need of a wheelchair at the age of 10 years. The life expectancy has also been significantly extended through the use of corticosteroid treatment and higher standards of medical care, such as non-invasive ventilation, although patients still tend to die from cardiac and respiratory complications [13]. DMD patients develop a severe cardiomyopathy that generally manifests at about 10 years and is prevalent in most patients by 20 years of age [14]. So, the above data demonstrated that we have to concentrate our efforts on early detection of DMD in order to optimise attempts to use a disease modification therapeutic approach.

The earlier the age of onset, the better the diagnostic opportunities and further the feasibility of specified therapy [15].

Furthermore, the lack of information on the level of CK in the initial stages or during the course of the disease on some of the patient’s records, indicates an underestimation of this laboratory feature in the cases of the DMD. Moreover, in some cases elevated CK level was misdiagnosed as hepatitis and patients were wasting time finding the correct diagnosis [16].

The number of involved patients is 84, which is not high compared with other nationwide studies. The percentage of 58,3%in large deletions by MLPA in our study is not as high as in Bladen’s paper of the TREAT-NMD DMD Global database (80%). [17] However it is close to the Italian cohort of 1162 DMD patients, where deletions were found in 57%of cases [18]. In correlation to a study in an Asian country, China (60.2%) the findings are similar [19(2)]. More often we have detected deletions in exons 48–52 in 12,7%, exons 48–50 and exons 45–52 and 49–50, respectively.

Nonsense mutations in our group of DMD males were found in 29,7%which is quite a high group of pathogenic variations. It was described that approximately 15%of DMD cases are caused by a nonsense mutation [5] and 17%of deletions are amenable for a target treatment approach. The previous studies in Kazakhstan in 2019 and 2021 demonstrated 27%among nonsense mutations in DMD gene [20, 21]. The highest level of nonsense mutations may be caused by excessive activity in seeking specified available therapy that is provided by pharmaceutical companies. So actively developed possibilities in disease course correction may lead to bias in analysis of statistical data in the country. This occurrence is more visible in low- or middle-income countries with no special programmes for disease screening due to restrictions in feasibility of free warranted genetic analysis. These statistical biases may lead to consequences within the whole medical care system, for example in terms of management (money planning for specified medicines).

Other countries do struggle with similar problems as Kazakhstani doctors: delayed diagnosis, funding deficits (22,23).

5CONCLUSION

CONFLICT OF INTEREST

The authors have no conflict of interest to report