The Xq22 inversion breakpoint interrupted a novel Ras-like GTPase gene in a patient with Duchenne muscular dystrophy and profound mental retardation

Abstract

A male patient with profound mental retardation, athetosis, nystagmus, and severe congenital hypotonia (Duchenne muscular dystrophy [DMD]) was previously shown to carry a pericentric inversion of the X chromosome, 46,Y,inv(X)(p21.2q22.2). His mother carried this inversion on one X allele. The patient's condition was originally misdiagnosed as cerebral palsy, and only later was it diagnosed as DMD. Because the DMD gene is located at Xp21.2, which is one breakpoint of the inv(X), and because its defects are rarely associated with severe mental retardation, the other clinical features of this patient were deemed likely to be associated with the opposite breakpoint at Xq22. Our precise molecular-cytogenetic characterization of both breakpoints revealed three catastrophic genetic events that had probably influenced neuromuscular and cognitive development: deletion of part of the DMD gene at Xp21.2, duplication of the human proteolipid protein gene (PLP) at Xq22.2, and disruption of a novel gene. The latter sequence, showing a high degree of homology to the Sec4 gene of yeast, encoded a putative small guanine-protein, Ras-like GTPase that we have termed "RLGP." Immunocytochemistry located RLGP at mitochondria. We speculate that disruption of RLGP was responsible for the patient's profound mental retardation.

Figure 1

Results of investigations of Xp21. A, Summarized genomic structure at Xp21 around one breakpoint in the patient with inv(X). YACs and STSs between markers WI-7235 and WI-5231 were positioned according to information archived in the Whitehead Institute/MIT Center and Sanger Centre databases. Black, hatched, and white bars indicate YACs that showed no specific signal, reduced signal, or normal signal, respectively. The presence (+) or absence (–) of a PCR product from the STS markers is indicated in the third row of the top panel. The presence (+) or absence (–) of PCR products is indicated in the third row of the bottom panel. E = EcoRI; H = HindIII; B = BglII. B, FISH analysis, using a BAC that contains human PLP sequence. In addition to a normal copy of PLP at Xq22, an extra-copy signal was detected at Xp21 on inv(X) in both the patient and his mother. C, Southern blot analysis, using the Pr3 product as a probe. Lane P, DNA from patient with inv(X)(p21.2q22.2); lane M, patient's mother’s DNA; and lane N, normal DNA. Rearranged fragments, which appeared when the DNA samples of the patient and his mother were digested with HindIII (9.8 kb), HindIII/EcoRI (4.8 kb), or HindIII/BglII (2.6 kb), are indicated by asterisks (*). D, Genomic sequence spanning the inversion breakpoint. Clone c11T3 was isolated from a cosmid library constructed from DNAs of the patient’s LCLs.

Figure 1

Results of investigations of Xp21. A, Summarized genomic structure at Xp21 around one breakpoint in the patient with inv(X). YACs and STSs between markers WI-7235 and WI-5231 were positioned according to information archived in the Whitehead Institute/MIT Center and Sanger Centre databases. Black, hatched, and white bars indicate YACs that showed no specific signal, reduced signal, or normal signal, respectively. The presence (+) or absence (–) of a PCR product from the STS markers is indicated in the third row of the top panel. The presence (+) or absence (–) of PCR products is indicated in the third row of the bottom panel. E = EcoRI; H = HindIII; B = BglII. B, FISH analysis, using a BAC that contains human PLP sequence. In addition to a normal copy of PLP at Xq22, an extra-copy signal was detected at Xp21 on inv(X) in both the patient and his mother. C, Southern blot analysis, using the Pr3 product as a probe. Lane P, DNA from patient with inv(X)(p21.2q22.2); lane M, patient's mother’s DNA; and lane N, normal DNA. Rearranged fragments, which appeared when the DNA samples of the patient and his mother were digested with HindIII (9.8 kb), HindIII/EcoRI (4.8 kb), or HindIII/BglII (2.6 kb), are indicated by asterisks (*). D, Genomic sequence spanning the inversion breakpoint. Clone c11T3 was isolated from a cosmid library constructed from DNAs of the patient’s LCLs.

Figure 2

Results of investigations of RLGP. A, Genomic structure of the RLGP gene in relation to the breakpoint located in its 5′ region. B, The nucleotide and deduced amino acid sequences of the RLGP-encoding cDNA. The nucleotide sequence is numbered on the left, and the deduced amino acid sequence is numbered on the right. The ras-like GTPase domain is boxed, and an SOCS domain is underlined. The amino acids that are not identical to those in RAR2A are indicated in boldface type, and the amino acids in RAR2A are shown in parentheses.

Figure 3

Expression of RLGP mRNA in normal human tissues. The same blot was rehybridized with a GAPDH probe, as a control, for RNA loading and transfer.

Figure 4

Subcellular localization of RLGP. COS7 cells were transiently transfected with pEGFP-RLGP (A and D), fixed, and immunocytochemically stained using mouse monoclonal anti-human mitochondria (B) or anti-Grp78 (E) antibodies. Note that the localization of RLGP overlapped with antimitochondrial staining (C) but showed no specific accumulation in the endoplasmic-reticulum compartment labeled by anti-Grp78 (F).