Clinical and molecular characterization of a transmitted reciprocal translocation t(1;12)(p32.1;q21.3) in a family co-segregating with mental retardation, language delay, and microcephaly

Mental retardation (MR) is a childhood-onset neurodevelopmental disorder characterized by a reduced intellectual function that results in learning disability and impaired social adaptation. Approximately 2-3% of the general population is affected with MR; and males are more often affected than females [1]. Genetic defects including gross structural abnormalities of chromosomes, cryptic genomic rearrangements, and monogenic mutations are the leading cause of MR [2, 3]. Numerous genes with diversified biological functions have been found to be associated with syndromic and non-syndromic MR; moreover, most of the genetic mutations causing MR are rare, private mutations, indicating that the genetic etiology of MR comprises a variety of highly heterogeneous genetic defects [2, 3]. Despite the fact that many genes have been identified as being associated with MR, more MR genes remain to be discovered [2‐4].

Chromosomal rearrangements associated with MR may provide an opportunity to discover novel genes associated with this condition. Chromosomal translocations may lead to clinical phenotypes via direct gene disruption, formation of chimera genes, or alteration of the expression of genes near the breakpoint via the position effect [5‐7]. Several MR-associated genes have been discovered through mapping of the breakpoints of chromosomal translocations, such as the dedicator of cytokinesis 8 gene (DOCK8) at 9p24 [8]; the potassium large conductance calcium-activated channel, subfamily M, alpha member 1 gene (KCNMA1) at 10q22.3 [9]; the autism susceptibility candidate 2 gene (AUTS2) at 7q11.2 [10]; the oligophrenin 1 gene (OPHN1) at Xq12 [11]; the Cdc42 guanine nucleotide exchange factor (GEF) 9 (ARHGEF9) at Xq11.1 [12]; and the reelin gene (RELN) at 7q22 [13].

As part of serial genetic studies of mental retardation, we detected a reciprocal translocation between chromosome 1p and 12q in the karyotype analysis of a family affected with severe MR, language delay and microcephaly. The translocation was transmitted from the mother to her two boys and co-segregated with the phenotypes. Herein we report the clinical phenotypes and the molecular characterization of the translocation associated with the phenotypes in this family.

The Taiwanese family was ascertained through the psychiatric clinic of Tzu-Chi General Hospital, Hualien, Taiwan. The family received medical attention due to the psychomotor retardation of the eldest boy of the family. All family members gave their written consent after all the details of the study were fully explained.

In the present study, we successfully mapped the breakpoint sequences of a reciprocal translocation between chromosome 1 and 12 that co-segregated with mental retardation, language delay, and microcephaly in a two-generation family. The breakpoint on chromosome 1 did not disrupt any known gene directly. The genes closest to this breakpoint included the CYP2J2 gene located ~ 621 Kb centromeric from the breakpoint, and the NFIA gene located ~ 529 kb telomeric from the breakpoint. The chromosome 12 breakpoint did not directly disrupt any known gene, either. The breakpoint was located ~ 3.4 kb downstream of the 3' UTR of the ALX1 gene, which was the gene closest to the breakpoint. The ALX1 gene encodes the aristaless-like homeobox 1 transcription factor that is involved in craniofacial development. Disruption of the ALX1 gene was recently reported to cause an autosomal recessive microphthalmia and a severe facial cleft [14]. In this study, ALX1 was considered as a putative candidate gene associated with the clinical symptoms of this family in view of its close location to the breakpoint and its biological function. We hypothesized that the expression of the ALX1 gene might be changed via the position effect by the translocation [7, 15, 16]. To test this hypothesis, we first attempted to measure the mRNA level of the ALX1 gene from lymphoblastoid cells in affected family members and control subjects using RT-qPCR; however, the mRNA level of the ALX1 gene was not detectable in the lymphoblastoid cells. To assess the influence of the translocation on ALX1 gene expression, we next conducted a reporter gene assay in Chinese ovary hamster (CHO) cells and a SKNSH neuroblastoma cell line. The results from this assay showed that the fusion construct between chromosome 12 and 1 had significantly higher reporter gene activity than the corresponding normal chromosome 12 sequences, indicating the fusion construct might enhance ALX1 gene expression.

The ALX1 gene, also known as the CART1 gene, encodes a 326-amino acid cartilage paired-class homeoprotein 1 that acts as a transcription factor and binds to palindromic sequences consisting of two TAAT separated by 3 or 4 base pairs [17]. In rodents, the Alx1 gene is expressed in forebrain mesenchymal cells, branchial arches, limb buds, and chondrocytes during embryogenesis [18, 19]. Homozygous Alx1 gene knockout mice did not show bone abnormalities, instead, they showed defects in neural tube closures that resulted in acrania, meroanencephaly, and other head abnormalities [19], while the heterozygous Alx1 knockout mice were apparently normal [19]. Recently, Uz and colleagues reported the first human clinical phenotypes associated with ALX1 gene mutation in two families [14]. They found that complete loss of the ALX1 function in these two families seriously disturbed early craniofacial development and resulted in severe frontofacionasal dysplasia, including bilateral extreme microphthalmia, severe facial cleft, complete palate cleft, and low-set posteriorly rotated ears; the frontofacionasal dysplasia in these two families presented in a recessive mode of inheritance. In one family, the affected family members showed a homozygous 3.7 Mb deletion containing the ALX1 gene. In the other family, a homozygous G-to-A mutation at the donor-splicing site of intron 2 was identified in the affected patient. The clinical symptoms caused by the loss-of-function mutations of the ALX1 gene were related to the clinical spectrum of frontofacionasal dysplasia caused by the two other ALX genes, ALX3 and ALX4. Patients with homozygous mutations in the ALX3 gene presented distinctive clinical features of frontofacionasal malformations, including hypertelorism, wide nasal bridge, bifid nasal tip, broad columella, widely separate slit-like nares, long philtrum with prominent bilateral swellings, and midline notch in the upper lip and alveolus, termed frontorhiny [20]. Homozygous mutation in the ALX4 gene was found to be associated with frontofacionasal dystosis syndrome, characterized by total alopecia, a large skull defect, coronal craniosynostosis, hypertelorism, a severely depressed nasal bridge and ridge, a bifid nasal tip, hypogonadism, callosal body agenesis, and mental retardation [21]. Together, these findings suggest all three ALX family genes have an essential, non-redundant role in the craniofacial development in humans, and the loss-of-function mutations of these genes may cause severe cranio-facial-nasal abnormalities.

Based on the reporter gene assay, we speculate that the clinical phenotypes of the family in the present study including severe MR, language delay, and microcephaly are likely to be associated with over-expression of the ALX1 gene, which contrasts with the clinical phenotypes of the loss-of-function mutation of the ALX1 gene as reported recently [14]. The pathogenesis of the over-expression of the ALX1 gene needs further investigation. A previous study reported that the homodimerization of the Alx1 is necessary for its transcription activity [22]. In their study, the authors isolated two additional isoforms of the Alx1 that were generated by alternative splicing from rat chondrosarcoma. These two additional isoforms competed with the wild-type Alx1, exerting a dominant negative effect. Brouwer et al. also reported that expression of mutant Alx1 cDNA with the deletion of the aristaless-domain resulted in severe cranial and vertebral abnormalities, supporting a dominant negative role or gain-of-function of certain mutations in the Alx1 gene [23]. Hence, we speculate that the over-expression of ALX1 might have a dominant negative or gain-of-function effect and result in the clinical phenotypes of this family. The clinical features associated with the over-expression of the ALX1 gene have not yet been reported in the literature; the family reported in the present study might be the first to indicate that increased expression of the ALX1 gene may lead to microcephaly, language delay, and mental retardation. Our findings may expand the clinical spectrum of ALX-related gene mutations in humans.

Notably, the association between the translocation and the clinical presentations in this family should be interpreted with caution. As an alternative, it is likely that the concurrence of the translocation in the affected family members might be due just to chance. In addition, the maternal grandmother of this family was reported to have mental retardation too; and the affected mother had a younger brother who was reported to have normal intelligence. However, neither of them was available for investigation of the origin of the translocation, which is a limitation of this study.

We identified a reciprocal translocation t(1;12)(p32.1;q21.3) that co-segregates with microcephaly, language delay, and mental retardation in a two-generation family. The mapping and characterization of the breakpoint sequences suggest that the translocation may enhance the gene expression of the ALX1 via the position effect and may thus lead to clinical phenotypes of this family.