Identification and analysis of deletion thresholds in four patients with Mohr-Tranebjærg syndrome (MTS)

Breakpoints identified

In all four cases, deletion thresholds were identified by a combination of successive PCR tests for the presence or absence of amplified products. Subsequently, mate-pair sequencing was performed to enable precise primer placement for successful PCRs spanning the breakpoints (Fig. 1).

Figure 1

Overview of the TIMM8A and BTK gene region (hg19 chrX: 100, 561, 900–100, 630, 700) with custom lanes showing deleted regions from cases 1-4, marked with red horizontal bars. The RefSeq gene track is displayed in blue, and below repeating elements (long terminal repeats (LTRs), short and long interleaved elements (SINE and LINES, respectively)) are displayed based on information from RepeatMasker. For each of the four cases, at least one of the junctional deletion regions was located in repeating elements.

Case 1 (XLA-MTS)

Australian male patient, briefly mentioned in Tranebjærg, 2013, as patient 33 (18 months) in Table 15. Clinically, the boy presented with profound hearing loss and had a cochlear implant (CI), as well as severe behavioral abnormalities and XLA. At 16, he was diagnosed with Autism Spectrum Disorder (Level 3), Attention Deficit Disorder, Dysgraphia. His academic performance was above the proficiency level with minimal support in earlier periods, but with a recent regression in his social-emotional development to that comparable to that of a preschooler. He suffered from considerable separation anxiety and was regularly seen by a child psychiatrist and treated for his psychiatric problems.

A 63.5 KB de novo deletion (ChrX: NC_000023.10:g.100564340_100627836del) covering TIMM8A and exon 4–19 of BTK was identified. Initially, the deletion was identified by sequential PCR, amplifying the genomic regions between TAF7L and BTK exon 1. Subsequently, partner pair sequencing and finally PCR spanning breakpoints were used to delineate deletion breakpoints. Both breakpoints were located in the Alu SINE (Short Interspersed Elements) repeat regions. The proband’s mother did not harbor the deletion in DNA purified from blood, indicating a de novo origin.

Case 2 (XLA-MTS)

Scottish male patient, born 2000, briefly mentioned in Tranebjærg, 2013, as patient 34 in Table 15. Clinically, the boy presented with deafness and some behavioral abnormalities, as well as XLA. No visual abnormality was described at the age of 9 years. At 21, he suffered from chronic lung disease, bronchiectasis and mild intellectual disability with some behavioral problems, including outbursts of aggression and difficult relationships with his peers at school. Speech delay at 18 months of age was initially attributed to repeated ear infections, but sensorineural hearing loss was later diagnosed and treated with hearing aids as he did not meet the criteria for CI before he was born. 6 years old, when he had a left side CI. Around the same time, he had learned sign language, which he tends to prefer as a mode of communication. There is no suggestion of neurological, dystonic or visual problems. Current schooling is in the college education of craftsman skills.

Using array-CGH, ​​de novo deletion of 17-19 kb with breakpoints in intron 1 of TIMM8A and intron 5 of BTK has been detected. However, PCR spanning breakpoints, based on the array-CGH result, was not successful. As a result, through partner pair sequencing and PCR covering breakpoints, a larger rearrangement was detected, involving a deletion of 39.9 kb (ChrX: NC_000023.10: g.100580141_100619992del) including the entire coding region of TIMM8A and exon 6–19 of BTK. Additionally, a 59 bp reverse insertion was observed in the breakpoint region (Fig. 2). The insertion corresponded to a region, mapped to chrX:100, 582, 138–100, 582, 196, which was located in the deleted sequence. The proximal breakpoint of the deletion was located in a LINE (long intercalated element) and the distal breakpoint was located in a SINE. The two breakpoints of the 59 bp insertion were located in a LINE.

Figure 2
Figure 2

Breakpoint junctions and flanking regions identified in cases 1-4. (a) Sequence chromatograms are displayed with the region spanning the breakpoint identified above. Regions of microhomology are indicated in black boxes for cases 1, 3, and 4. However, it is not possible to determine which end of the overlap region is the true breakpoint. The 59 bp inverted insertion identified in the breakpoint region in case 2 is underlined. (b) Sequences of breakpoint junctions identified in cases 1-4 relative to the upstream and downstream reference sequence. Regions of microhomology are shown in gray.

Case 3 (XLA-MTS)

Italian male patient, previously released in 200416, presenting clinically with severe hearing impairment from age 2, a marked reduction in visual acuity, XLA, and later in life dystonia developed. Subsequent follow-up investigations revealed a biased X-inactivation pattern in the mother, who also had clinical symptoms including dystonia. Since the initial report, the patient and his mother have both died. Pizzuti et al., by successive PCR, demonstrated that the deletion included the two exons of TIMM8A and a partial removal of BTK (the telomeric breakpoint was located between BTK exons 7 and 19)16. We performed further successive PCR tests, which demonstrated that BTK exon 15 and the first 788 base pairs of intron 16 are intact, while exon 16 could not be amplified. Finally, using partner pair sequencing and breakpoint spanning PCR, a deletion of 27.2 kb (chrX: NC_000023.10: g.100582893_100610076del) including the entire coding region of TIMM8A and exon 16–19 of BTK was identified. The breakpoints were both located in SINEs. The deletion was maternally inherited as evidenced by PCR and breakpoint sequencing.

Case 4 (MTS)

Dutch male patient, born 1986, briefly mentioned in Tranebjærg, 2013, as patient 35 in Table 15. Clinically, the boy presented with congenital deafness, an abnormal VEP (visual evoked potential) response and reduced visual acuity, as well as progressive ataxia. No dystonia was observed. By successive PCR a deletion of 2.1 kb (chrX: NC_000023.10:g.100600271_100602381del) covering exon 2 of TIMM8A was identified. The telomeric part of the deletion was located in a SINE. As evidenced by PCR and breakpoint sequencing, the deletion was inherited from the mother and the affected brother of the mother also carried the deletion. The grandmother, having an affected son and a carrier daughter, must be a mandatory carrier; however, the deletion could not be detected by PCR of DNA purified from blood, suggesting germline mosaicism.

Checking identified breakpoints

For each of the four cases, breakpoints were validated at base pair resolution, and all junctions were confirmed by Sanger sequencing. In cases 1, 3 and 4, junctional microhomology was observed, ranging from 2 to 38 nucleotides. In case 2, an inverted insertion of 59 bp was observed in the junctional region, corresponding to position chrX: 100, 582, 138–100, 582, 196 (Fig. 2).

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