Q14.2

[Laszlo Perry]

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Split hand-split foot malformation (SHFM; OMIM 225300) or ectrodactyly is a congenital defect of digit formation characterized by aplasia of the central digits with fusion of the remaining digits. Most cases are isolated with autosomal-dominant inheritance and incomplete penetrance, but some pedigrees with autosomal-recessive or even X-linked forms have also been reported (reviewed by Duijf et al1). Ectrodactyly also occurs in several syndromes and can be found associated with a variety of other developmental anomalies.

Ectrodactyly has been repeatedly reported in association with a variety of other developmental anomalies. As an example, a holoprosencephaly, hypertelorism and ectrodactyly syndrome (HHES), considered as an independent clinical entity, was reported to be associated with an apparently balanced de novo translocation [t(2;4)(q14.2;q35)] in a Mexican patient.8 In addition to this case, an association of ectrodactyly with holoprosencephaly has been reported in six additional probands (reviewed by Knig et al9).

In this study, we characterize the breakpoints of a de novo apparently balanced chromosomal translocation [t(2;11)(q14.2;q14.2)] associated with bilateral split foot malformation (SFM) identified in a fetus during ultrasound prenatal screening. Furthermore, we also mapped the chromosome 2 breakpoint identified in the previously reported Mexican proband with HHES.

The probands are described in the Results section. A group of 35 unrelated patients with either typical, atypical, isolated or syndromic forms of SHFM and a control group of about 100 unrelated individuals were used for mutation screening of the candidate genes.

Following informed consent from the parents, fetal blood sample was obtained after voluntary termination of pregnancy from the umbilical vein and a lymphoblastoid cell line (LCL) was established. Bacterial artificial chromosome (BAC) clones were obtained from the Sanger Institute (Hinxton, Cambridge, UK) and the BACPAC Resources Center at Children's Hospital Oakland Research Institute (Oakland, CA, USA).

Genomic DNA was prepared from peripheral blood lymphocytes and LCLs by standard molecular techniques. LCL or DNA samples were unavailable from the Mexican proband with HHES. BAC DNA was extracted by the alkaline lysis method performed according to the protocol from the Hubbard Center for Genome Studies ( ).

Metaphase and prometaphase chromosome preparations were obtained from LCLs and phytohemagglutinin-stimulated lymphocytes by standard cytogenetic techniques. Cytogenetic analysis was performed on conventional Trypsin-Leishman G-banded metaphases. Only three chromosome slides were available from the proband with HHES.

Screening of the control group and additional family members for the presence of the identified potential pathogenic mutations was carried out by single-stranded conformation polymorphism analysis or restriction enzyme cleavage and direct sequencing, performed as described earlier.13, 14 The screening conditions are also available on request.

Cytogenetic analysis performed from cultured amniotic fluid cells of the fetus revealed a de novo apparently balanced reciprocal chromosomal translocation [t(2;11)(q14.2;q14.2)] (Figure 1b). Both parents were cytogenetically normal. Larger genomic imbalances were ruled out on the basis of chromosome CGH (data not shown), and the reciprocity of the translocation was confirmed by FISH analysis (Figure 1c).

Schematic representation of the chromosome 2 breakpoint region associated with isolated SFM. (a) Schematic ideogram of chromosome 2; the breakpoint region is highlighted by a box. (b) Physical map across the chromosome 2 breakpoint region. Some of the STS markers from this region, together with the GeneBank sequence acc. no. of BACs spanning this region, are noted above the map. Location and genomic organization of the candidate genes are indicated. Numbered vertical lines indicate exons. (c) Detailed map of the breakpoint region indicating the position of evolutionarily conserved sequence elements and of CpG islands. Arrow indicates the location of the breakpoint.

SYBR green-based QRT-PCR analysis revealed that the expression level of INHBB and GLI2 in eight control LCLs was barely detectable and too low to allow reliable quantification. In patient cells, the expression of both genes was low as well, but more readily detectable than in the control LCLs, indicating that, if anything, expression of these is increased rather than decreased.

Activins or inhibins are best known for their endocrine role in pituitary follicle-stimulating hormone synthesis and secretion and for their reproductive function. Besides these functions, Merino et al23 very convincingly showed an important role of activin/inhibin signalling in digital skeletogenesis. They showed that exogenous implantation in the interdigital mesoderm of chick embryos of various dimers of activins leads to extra digit formation, whereas follistatin, a natural antagonist of these, blocks activin-induced or physiological digit formation. Furthermore, they also showed the interaction of activins with BMPs. Despite this, no major anomalies were observed in homozygous null mutant mice for activin-βB other than the failure of eyelid fusion.24

The identification of a pathogenic mutation within one of these candidate genes, in non-translocation patients presenting similar congenital anomalies, would unequivocally implicate this gene in the aetiology of the corresponding malformation. Several unreported mutations have been identified in introns or in UTRs of the candidate genes. All of these were evaluated as potential cryptic splice sites or branch point sites. Presently, on the basis of our current knowledge, none of these can be considered as a pathogenic mutation. Among the identified amino-acid substitutions, the most important is the double mutation (c.4558G>A, Asp1520Asn and c. 4054A>G, Met1352Val) in the GLI2 gene of an isolated patient with nonsyndromic SHFM. The high level of conservation of residue Asp1520 (Supplementary Table) suggests that this double mutation may well be pathogenic, but surprisingly, this was also identified in the control group and was inherited from a phenotypically normal mother. Loss-of-function mutations in GLI2 have been associated with a phenotype of defective anterior pituitary formation, pan-hypopituitarism and holoprosencephaly-like mid-facial hypoplasia.16

The fact that no clear pathogenic mutation was identified in these groups of patients (present study and Babbs et al11) and that QRT-PCR did not show any evidence for downregulation of INHBB and GLI2 may reflect that the molecular alterations introduced by the translocations may have very specific effects in terms of quantitative spatiotemporal expression of one or more genes from the breakpoint regions. It is very likely that such effects may not be mimicked by intragenic gene mutations.

One possibility is that all breakpoints affect the developmental expression of one or more genes from this region by disruption of the architecture of their corresponding long-range control elements. In this scenario, long-range control elements must be operational at very long distances from the target site, given the large separation of the two translocation breakpoints. The alternative possibility is that positional effects introduced by the translocations lead to epigenetic alterations of the local chromatin structure, for example by inappropriate DNA methylation or histone modifications. Both possible mechanisms are predicted to result in quantitative spatiotemporal disrupted expression of one or several genes during embryonic development.

The very low expression levels of the candidate genes INHBB and GLI2 in lymphoblast cell lines prohibited their quantitative analysis for their possible involvement. It is very likely that such studies can be reliably performed only by using material from tissues obtained from appropriate developmental stages. Therefore, further research should focus on the identification of long-range regulatory elements that control transcription of genes in the region encompassing the breakpoints. Such elements may be binding sites for transcription factors such as TP63, which is mutated in a number of syndromes that have SHFM as a major hallmark. Ultimately, the identification of specific functional mutations in such elements, such as microdeletions and nucleotide changes, in SHFM patients has to prove the functionality of such elements in health and disease.

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We report on the case of a pregnant woman with hyposomia who was previously suspected of having Turner syndrome. Prenatal cytogenetic diagnostics showed a fetal karyotype of 46,XX,dup(13)(q14.2q21.1) ish.13q14(RB1 x 3). Parental and grandparental chromosome analyses were performed and the dup(13) was found to be of maternal origin (de novo). The pregnancy was continued and a healthy female child was born with normal development apart from growth retardation. The reported chromosomal aberration is, together with two other cases reported in the literature, the first hint of a short stature-like phenotype due to dup(13)(q14.2q14.3).

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