Identification of GAA variants through whole exome sequencing targeted to a cohort of 606 patients with unexplained limb-girdle muscle weakness

Pompe disease (OMIM 232300) is a rare autosomal recessive lysosomal storage disorder that most prominently affects muscle tissue. The disease is generally classified into two broad categories: an infantile- and a late- onset form. Patients with the infantile-onset form of the disease typically present with generalised weakness, hypotonia, respiratory distress and cardiomyopathy, and without intervention do not survive beyond 12 months of age [1]. Late-onset Pompe disease is more clinically heterogeneous [2], yet often displays a central characteristic manifestation of a slowly progressive proximal myopathy. This occurs with respiratory weakness and elevated serum creatine kinase activity, while there is normally no clinically relevant cardiac involvement [3]. Estimates of the frequency of all cases of Pompe disease vary, but have been reported to be as high as approximately 1:40,000 [4, 5]. The late-onset classification of the disease is even rarer at a frequency of 1:57,000 [5].

The varied clinical spectrum can be attributed to the many different genetic mutations that are associated with Pompe disease; to date, the associations of over 522 variants have been reported and collated by the Pompe Disease Mutation Database [6]. Such mutations occur within the GAA gene and result in a deficiency of the encoded lysosomal enzyme, acid α-glucosidase, which is essential for glycogen hydrolysis. The accumulation of glycogen within lysosomes subsequently impairs the correct functioning of the organelles and the affected tissue, primarily skeletal and cardiac muscle [7], and results in the clinical presentation of Pompe disease.

Considering the rarity and variability of the disorder, a correct clinical diagnosis is often difficult to achieve, and so many patients are therefore not treated with an efficacious disease-modifying enzyme replacement therapy (ERT) in a timely manner. ERT, a recombinant human acid α-glucosidase termed alglucosidase alfa, has been reported to extend survival in infantile Pompe disease [8] and ameliorate disease progression of the late-onset form [9]. As prompt diagnosis and treatment is beneficial to patient survival [10], a more robust investigatory approach is required to detect and diagnose affected individuals in the early stages of the disease.

Exome sequencing is a useful tool to interrogate the proportion of the genome that is enriched for functional coding variants, specifically those that are able to disrupt protein structure and function [11]. This unbiased analysis enables the detection of distinct mutations in patients with overlapping phenotypes, overall expanding existing genotype-phenotype correlations. Exome sequencing, therefore, has the benefit of furthering the understanding of disease pathology, offering accurate diagnoses where the traditional methodologies of clinical examinations failed to do so, and in some cases also enabling a prompt intervention in the disease progression.

The MYO-SEQ project was established in 2014 and aimed to use whole exome sequencing to (i) contribute to the diagnostic pathway of patients affected by limb-girdle muscular weakness, (ii) improve the diagnostic awareness of rare genetic neuromuscular diseases, and (iii) speed up the integration of next-generation sequencing technologies into healthcare [12]. We screened 606 patients with unexplained limb-girdle weakness for potentially pathogenic variants in 169 genes that are known to be associated with muscle disease. Here, we report on the characterisation of twelve European patients (eight index cases and four siblings) who harboured disease-associated variants within GAA as identified through the MYO-SEQ project.

Whole exome sequencing is emerging as an affordable technology to investigate rare, monogenic diseases. Coding and functional regions account for only 1% of the entire human genome, yet harbour 85% of known disease-causing variants [25]. On this basis, we sequenced the exomes of a cohort of 606 patients with unexplained limb-girdle weakness. We examined a panel of 169 genes that were known to be associated with muscle diseases with Mendelian patterns of inheritance in a large cohort of European patients with unexplained limb-girdle weakness. Overall, we identified eight unrelated index cases and four siblings that had compound heterozygous mutations in GAA: these patients were likely to be affected by Pompe disease, a rare lysosomal storage disorder that is commonly characterised by proximal muscle weakness [2]. The remaining 168 genes are currently under analysis and we are yielding similarly positive results for many other muscle diseases; so far the overall diagnostic rate for the project is 49%.

We have shown that next-generation sequencing is advantageous in the healthcare and diagnosis of patients suffering from unexplained limb-girdle muscle weakness. Other studies have shown similar benefits [26], achieving a correct diagnosis for patients with overlapping, and therefore indistinguishable, clinical phenotypes [27]. In a recent publication that interrogated the exomes of 504 patients of European descent, variants in GAA were considered pathogenic in 10 patients (1.9% of the cohort) [28]. This is highly comparable to the 12 patients with GAA variants detected by MYO-SEQ. While such an approach helps better understand rare neuromuscular disorders, it was especially important in this study for the patients that suffered from treatable conditions such as Pompe disease. It is most beneficial to the affected individuals that ERT is initiated as soon as possible as the therapy acts to ameliorate disease progression [29]. Despite DBS tests being widely available, inexpensive and sensitive to changes specifically in α-glucosidase activity [30], they can sometimes fail to robustly detect subtle changes in enzyme activity. This is considered more common in later onset forms of the disorder, where the level of enzyme activity may be proportional to the age of onset [29]. Although reduced enzymatic activity was detected by DBS tests in 7.6% of patients in a cohort presenting with elevated serum creatine kinase and/or limb-girdle muscular weakness, late-onset Pompe disease was only confirmed in 2.4% of the patients [31]. DBS tests may therefore be inconclusive for many patients with a proximal muscle weakness phenotype. Such a discrepancy was exemplified in the clinical assessment of patient 18 who presented with the onset of symptoms in her fifth decade of life and normal to slightly lower α-glucosidase activity. Despite the borderline enzymatic activity levels detected, the individual was subsequently found to carry two reported pathogenic GAA variants. Therefore, exome sequencing offers an alternative, accurate and reliable methodology for rare disease diagnosis where traditional detection techniques may not be as efficient. We suggest that DBS should still be used as a first-tier diagnostic step when Pompe disease is suspected, however, diagnostic yields are influenced by the type and level of pre-screening, and so it is inaccurate to compare the outcomes of different approaches.

As Pompe disease is an autosomal recessive disorder, single heterozygous variants are unlikely to result in a depletion of α-glucosidase activity that is sufficient to cause a clinical phenotype. Accordingly, it has been found that many affected individuals carry compound heterozygous mutations rather than isolated heterozygous or homozygous variants [32‐35]. This immediately allowed our analysis to be focussed on the patients that harboured two variants: of course, a second variant may still be missed if it resides in a deep intronic region not covered by WES. Nevertheless, of the nine compound heterozygous patients that we identified, we considered eight to carry variants that were sufficiently severe to cause Pompe disease. Four of the ten likely pathogenic variants that were identified in this study had never been previously reported in Pompe disease [6] and did not occur in the ExAC control population [14]. These variants that we present here – c.1192delC, c.2020C > G, c.2051C > G and c.2716G > A – are therefore novel in the understanding of Pompe disease.

As would be expected based on previous findings [23, 35], the intronic c.-32-13 T > G variant was the most frequent in this largely Caucasian population, and occurred in six compound heterozygous index cases. It is therefore essential that analyses of GAA gene sequences should be extended to flanking regions in order to capture such pathogenic intronic variants. This variant affects a splice site of the gene, and so the result is the production of alternative transcript isoforms and low levels of α-glucosidase activity. This can give rise to the typical spectrum of Pompe disease phenotypes depending on the haplotype it occurs in [36]. Overall, the specific mutations, their haplotypes, and their genetic positions are all key in determining enzymatic activity and thus, the clinical phenotype of patients affected by Pompe disease.