Dear Editor,
We appreciate all the comments and remarks made by Josef Finsterer [1] regarding our article, Co-occurrence of CAPN3 homozygous mutation and CCTG expansion in the CNBP gene in a patient with muscular dystrophy [2]. We agree with the possible limitations of the study, such as the lack of determination of the exact number of CCTG repeats, using techniques such as Southern blotting. However, several concerns need clarification regarding why most laboratories do not routinely report the exact pathological number of repeats in myotonic dystrophy type 2 (DM2).
For instance, the region of (CCTG)n in DM2 is part of a complex and variable motif containing (TG)n(TCTG)n(CCTG)n repeats, which can be determined by sequencing. However, the pathological number of (CCTG)n repeats ranges from approximately 75 to over 11,000 units [3]. This heterogeneity, along with the large number of CCTG repeats, means that some techniques have technical limitations. For example, triplet-repeat primed (TP)-PCR and quadruplet-repeat primed (QP)-PCR can detect expansions in all size ranges but do not provide information about the exact length of the expanded repeat due to signal extinction in the higher size regions [3]. On the other hand, Southern blotting may generate false positive (or false negative) results due to somatic instability and cross-hybridization of the probe [3].
According to best practice guidelines and recommendations on the molecular diagnosis of DM1 and DM2 in homozygous cases, repeat-primed PCR and/or Southern blotting of genomic DNA or long-range PCR products should be performed to detect possible repeat expansions [3]. Moreover, Catalli et al. [4], proposed that tetraplet-primed PCR (TP-PCR) should be a routine diagnostic tool, alongside short-range PCR, in the molecular diagnosis of DM2. Identifying the expansion is sufficient to confirm the clinical diagnosis of DM2, without the need to determine the exact number of CCTG repeats, especially since no clear correlation between the age of onset and expansion length has been demonstrated in DM2 [3].
Furthermore, to ensure reliability of genetic testing results, our laboratory undergoes annual external quality control assessments. Regarding the clinical picture of the proband, we also mentioned that, at this stage, the patient’s phenotype corresponds more closely with LGMDR1 than DM2. However, this may be attributed to the proband’s young age (20 years) [2]. In general, the average onset of DM2 is between 30 and 40 years of age [3].
The patient was first hospitalized at the age of 8, when a clinical diagnosis of Becker muscular dystrophy was made, but deletions and duplications in the DMD gene were ruled out. The patient was under specialized care and rehabilitation but no genetic diagnosis was established at the time. At the age of 16, he was referred for targeted next generation sequencing (NGS) testing with a clinical diagnosis of limb-girdle muscular dystrophy. At the age of 19, genetic testing for DM2 was also performed. Arriving at a final diagnosis was a lengthy process, as NGS, enabling massively parallel sequencing of many genes simultaneously, was implemented in our laboratory only in 2018. Given the complexity (presence of two disorders in a single patient) and the multisystemic nature of DM2, the patient continues to receive comprehensive, annual care from a multidisciplinary team of specialists.
Our goal was to highlight the possibility of co-occurrence of two distinct disorders (LGMDR1 and DM2) in a single patient, particularly in light of the relatively high prevalence of DM2 in Europe [5, 6].
