Detection of TRIM32 deletions in LGMD patients analyzed by a combined strategy of CGH array and massively parallel sequencing

Abstract

Defects in TRIM32 were reported in limb-girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathies (STM) and in Bardet-Biedl syndrome. Few cases have been described to date in LGMD2H/STM, but this gene is not systematically analysed because of the absence of specific signs and difficulties in protein analysis. By using high-throughput variants screening techniques, we identified variants in TRIM32 in two patients presenting nonspecific LGMD. We report the first case of total inactivation by homozygous deletion of the entire TRIM32 gene. Of interest, the deletion removes part of the ASTN2 gene, a large gene in which TRIM32 is nested. Despite the total TRIM32 gene inactivation, the patient does not present a more severe phenotype. However, he developed a mild progressive cognitive impairment that may be related to the loss of function of ASTN2 because association between ASTN2 heterozygous deletions and neurobehavioral disorders was previously reported. Regarding genomic characteristics at breakpoint of the deleted regions of TRIM32, we found a high density of repeated elements, suggesting a possible hotspot. These observations illustrate the importance of high-throughput technologies for identifying molecular defects in LGMD, confirm that total loss of function of TRIM32 is not associated with a specific phenotype and that TRIM32/ASTN2 inactivation could be associated with cognitive impairment.

Figure 1

(a and b) Family pedigree for patients 1 and 2.

Figure 2

TRIM32 variants identified (a) IGV viewer visualization of the c.1603delC frameshift mutation appearing as homozygous. (b) Sanger sequencing confirming the homogenous loss of a cytosine. (c) CGH analysis of chromosome 9 allowing the identification of a large deletion encompassing the whole TRIM32 gene in DNA from patient 1. The CGH mean log score value was −0.73 and the deletion covered 242 probes, corresponding to chr9.hg19:g.119447866_119572263del. CGH analysis is visualized with Signalmap software using the unaveraged option (1 × ). (d) CGH analysis of chromosome 9 allowing the identification of a large deletion encompassing the whole TRIM32 gene in DNA from patient 2. The CGH mean log score value was −2.23 and the deletion covered 260 probes, corresponding to chr9.hg19:g.119447810_119783902del.

Figure 3

(a) Analysis of boundaries. Upper panel: In red: primers used for definition of the boundaries. In light green: deletion of patient 1. In dark green: deletion of patient 2. The thinner lines correspond to area of uncertainties. Lower panel: zoom of the repeats present on the upper boundary region. (b) Putative consequences of the homozygous deletion of patient 2 on the ASTN2 protein. From top to bottom: the deletion and the WT ASTN2 protein (c) TRIM32 pathogenic variants reported in the literature and databases (http://www.lovd.nl/TRIM32). Modified from Cossée et al.