Mutations in JARID1C are associated with X-linked mental retardation, short stature and hyperreflexia

Abstract

Background: Mutations in the JARID1C (Jumonji AT-rich interactive domain 1C) gene were recently associated with X-linked mental retardation (XLMR). Mutations in this gene are reported to be one of the relatively more common causes of XLMR with a frequency of approximately 3% in males with proven or probable XLMR. The JARID1C protein functions as a histone 3 lysine 4 (H3K4) demethylase and is involved in the demethylation of H3K4me3 and H3K4me2.

Methods: Mutation analysis of the JARID1C gene was conducted in the following cohorts: probands from 23 XLMR families linked to Xp11.2, 92 males with mental retardation and short stature, and 172 probands from small XLMR families with no linkage information.

Results: Four novel mutations consisting of two missense mutations, p.A77T and p.V504M, and two frame shift mutations, p.E468fsX2 and p.R1481fsX9, were identified in males with mental retardation. Two of the mutations, p.V504M and p.E468fsX2, are located in the JmjC domain of the JARID1C gene where no previous mutations have been reported. Additional studies showed that the missense mutation, p.V504M, was a de novo event on the grandpaternal X chromosome of the family. Clinical findings of the nine affected males from the four different families included mental retardation (100%), short stature (55%), hyperreflexia (78%), seizures (33%) and aggressive behaviour (44%). The degree of mental retardation consisted of mild (25%), moderate (12%) and severe (63%).

Conclusion: Based on the clinical observations, male patients with mental retardation, short stature and hyperreflexia should be considered candidates for mutations in the JARID1C gene.

Figure 1

Mutation analysis of family K8545. (A) Partial pedigree of K8545. The arrow indicates the proband (III-1). Affected males are indicated by a black box and obligate carriers with a dotted circle. The circles with hatched lines indicate that these individuals have mental retardation and learning disability. The numbers below the female symbols indicate the X-inactivation status. (B) Sequence analysis of exon 3 of the JARID1C gene from a control individual and the proband (III-1) showing the G>A substitution (c.229G>A) which is highlighted in yellow. The amino acid change (p.A77T) is shown in blue. Intronic sequences are shown in lower case. (C) A 2% agarose gel electrophoresis of the allele specific polymerase chain reaction (AS-PCR) showing the segregation of the c.229G>A alteration. The 198 bp band was generated by primer sets (Ex3-MSPF/R) specific for the mutation. This band is associated with the mutation. The 310 bp band is generated by a primer set for a control gene (KIAA1111 Ex5F/R).

Figure 2

Mutation analysis of family K8835. (A) Haplotype analysis of family K8835 with microsatellite markers flanking the JARID1C gene. The boxed numbers indicate the haplotype shared among the individuals. The letters “P” and “M” indicate the paternal and the maternal allele, respectively. The haplotype analysis shows that the c.1510G>A mutation is a de novo event arising in the grandpaternal allele of individual II-2. The arrow indicates the proband. Affected males are indicated by a black box and obligate carriers with a dotted circle. The numbers in parentheses under the females indicate the X-inactivation status. (B) Sequence analysis of exon 11 of the JARID1C gene from a control individual and the proband (III-2) showing the G>A substitution (c.1510G>A). The G to A change is highlighted in yellow and the amino acid change (p.V540M) is shown in blue. (C) A 2% agarose gel electrophoresis of the BsaA1 restriction endonuclease digestion of amplified DNA fragments showing the segregation of the c.1510G>A alteration. The normal males have bands of 123 bp and 191 bp, whereas the affected males (III-2, III-4, and IV-1) have a single 314 bp band.

Figure 3

Mutation analysis of family K8140: (A) Pedigree of K8140 showing two affected brothers. The arrow indicates the proband (II-1). Obligate carriers are indicated with a dotted circle. The numbers below the females indicate the X-inactivation status. (B) Sequence analysis of exon 26 showing a two bp deletion (c.4441_4442delAG) which is highlighted in yellow. The amino acids in blue indicate five of the novel amino acids which result from the deletion before termination (p.R1418GfsX9). (C) DHPLC chromatogram showing the segregation of the deletion in the family.

Figure 4

Mutation analysis of family K9374. (A) Sequence analysis of exon 11 showing a G>A substitution (c.1583+5G>A) in intron 11 of the JARID1C gene. The G>A change is highlighted in yellow. The sequence from intron 11 is in lower case showing the exon/intron junction. (B) Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of K9374—lanes 1 and 4; affected male; lanes 2 and 5: control male; lanes 3 and 6: negative control (H₂O). Lanes 1 and 2 show the amplification of a 600 bp band from a control gene (ARHGEF9). Lane 4 shows the 429 bp band from the affected male which is lacking the exon 11 of the JARID1C gene, whereas in lane 5 there is normal amplification of a 611 bp band from the JARID1C gene spanning exons 9 to 13. (C) Sequence chromatogram showing the sequence across the exon10/exon12 junction of the abnormal transcript.

Figure 5

Partial amino acid sequence alignment of the JARID1C (ENSP00000364550) protein. Sequence alignment of the four members of JARID1 family of proteins (Hs_JARID1C, Hs_JARID1A, Hs_JARID1B and Hs_JARID1D) and the JARID1C orthologous proteins in Pan troglodytes (ENSPTRP00000038795), Mus musculus (ENSMUSP00000080814), Canis familiaris (ENSCAFP00000023835), Bos taurus (ENSBTAP00000019893), Xenopus tropicalis (ENSXETP00000015599), Danio rerio (ENSDARP00000040942), and Dm_lid (NP_723140). The missense mutations are indicated by the arrows and blue letters. Amino acids that differ from the JARID1C protein are shown in brown.