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Keywords:

  • brachydactyly;
  • multiple congenital anomaly syndrome;
  • postaxial polysyndactyly;
  • short stature;
  • X-linked mental retardation syndrome

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CLINICAL REPORT
  5. MATERIALS AND METHODS
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

The family observed in this study included affected males and asymptomatic females. The patients shared specific digital abnormalities including postaxial polydactyly, cutaneous syndactyly, and brachydactyly. In addition, the patients exhibited mild-to-moderate intellectual disability and short stature coupled with microbrachycephaly, scoliosis, and cerebellar and renal hypoplasia. No chromosomal alterations or copy number variations were found in the index case. The genetic linkage analysis, which focused on the X chromosome, and the haplotype analysis detected a ∼15.74 Mb candidate region located at Xp11.4–p11.21 with a LOD score of 4.8. Additionally, half of the mothers showed skewed X-inactivation, while the other mothers exhibited random inactivation patterns. The candidate region includes 28 protein-encoding genes that have not yet been implicated in human disorders. We speculate that the observed phenotype is compatible with a monogenic disorder in which the mutant gene plays a significant role during embryonic development. Based on the patients' clinical features, image studies, pedigree, chromosome location, and X-inactivation studies in the mothers, we propose that this family has a novel, specific syndrome with an X-linked recessive mode of inheritance. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CLINICAL REPORT
  5. MATERIALS AND METHODS
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Recently, the Online Mendelian Inheritance in Man (OMIM, http://www.ncbi.nlm.nih.gov/omim), Genatlas (http://www.genatlas.org), and Ensembl (http://www.ensembl.org) databases reported that mutations in 192/836 (23%) of the protein-encoding genes on the X chromosome account for 267 known phenotypes. While 135 other phenotypes have been mapped, an additional 125 phenotypes are hypothesized to be X-linked. Assuming that each of these phenotypes is attributable to mutations in single genes, it is theoretically possible that 38% of the true number of X-linked phenotypes have yet to be discovered.

Clinical examination and pedigree analysis can identify a known syndrome or a new entity. Furthermore, ascertaining of a new genetic entity will require of the establishment of an association between the phenotype and a gene mutation. The identification of a disease gene could be either a fast process if the mutation defines the exact location of the candidate gene or a labor-intensive process if a long list of genes in a candidate region is obtained after a linkage analysis.

In this study, we demonstrated the presence of a potentially new X-linked syndrome in a Mexican family; the syndrome is characterized by digital abnormalities, mild-to-moderate intellectual disability and short stature.

CLINICAL REPORT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CLINICAL REPORT
  5. MATERIALS AND METHODS
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Family Report

This study was approved by the ethics committee of the Facultad de Medicina, Universidad Autónoma de Nuevo León.

A five-generation Mexican family was studied (Fig. 1). Four of the eight affected males were clinically and molecularly examined. All patients had multiple congenital anomalies. No information was available on IV.22 or II.6–II.8 who were, according to family reports, also affected.

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Figure 1. Pedigree, genotyping, and haplotype and linkage analyses of the family. The positions of eight patients and obligate carrier females in the pedigree, which suggests an X-linked mode of inheritance of the phenotype, are indicated with standard genetic nomenclature. The genotypes for 18 markers at the pericentromeric region Xp21.3–q22.1 are listed below each analyzed patient; the shared haplotype between patients and their mothers is green. The cytogenetic location, identity, and LOD score results for each genetic marker are shown in the table (bottom right corner). The arrow indicates the index case. Relative position of the AR gene is indicated by an asterisk.

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The parents of these individuals were healthy with normal phenotypes; and although most of the family came from the same rural community (Ahualulco, México), consanguinity was ruled out after clinical interviews.

The affected individuals were highly sociable, cooperative, and physically active, but unemployed. We describe the clinical findings for each patient, beginning with the index case (IV.5), who was compared to all of the subsequently analyzed individuals.

The index case was the most severely affected patient in the family and had the greatest number of clinical features. He was the fifth child; in addition, his mother experienced three previous spontaneous abortions, including an affected male. This patient was born after an uneventful pregnancy. At birth, he had hypotonia, poor head control and feeding difficulties; he did not walk until he was 8 years old and spoke few words. At the time of this report, he is 42 years old and has mild-to-moderate intellectual disability. He communicates well using good sentence structure but possesses a limited vocabulary. On physical examination he had short stature (145 cm, <3rd centile), microbrachycephaly (53 cm, <3rd centile) and a low posterior hairline with a redundant scalp. His facial features included a narrow forehead, midface hypoplasia, arched eyebrows, right ptosis, epicanthic folds, convergent strabismus, nystagmus, a broad nose, a flat philtrum, thick lips, a high palate, gingival hyperplasia, microdontia, and low-set ears (Figs. 2A and 3A). His skeletal abnormalities included cubitus valgus, scoliosis and limb malformations. He had bilateral postaxial polydactyly type B (PAP-B), bilateral brachydactyly of the 2nd, 4th, and 5th fingers and bilateral complete cutaneous webbing of the 4th and 5th fingers (type 3) with camptodactyly (Figs. 2B and 3B) of the hands. Foot abnormalities included bilateral cutaneous webbing of the 2nd and 3rd toes (type 5) and right postaxial polydactyly type A (PAP-A; Figs. 2C and 3C).

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Figure 2. Abnormal external phenotypic features of the four patients. A lateral facial view shows microbrachycephaly, low-set ears and low posterior hairlines are present in all patients (A,D,G,J). Patients V.12 and V.17 (H,K) showed incomplete syndactyly revealing brachydactyly as a total or partial absence of distal interphalangeal creases in the 2nd, 4th, and 5th fingers. This event was likely masked in IV.5 and IV.17 due to the presence of complete syndactyly combined with camptodactyly (B,E). Similarly, the bilateral postaxial polydactyly in the hands and webbing of the 4th and 5th fingers were more evident in generation IV (B,E) compared with generation V (H,K). In contrast, the postaxial polydactyly of the feet was similar in both generations and was either right (C,F,I) or left (L). A bilateral duplication of the distal phalanx of the left 4th toe was only present in generation IV (C,F). A bifid nail in the 3rd left toe was detected in V.12 (I). The extra digits of V.17 were surgically removed at infancy.

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Figure 3. Facial and digital abnormalities; and image studies of the index case. A: A front view of the face showing a narrow forehead, midface hypoplasia, arched eyebrows, right ptosis, epicanthic folds, a broad nose, flat philtrum, thick lips and low-set ears. B: The hands show bilateral postaxial polydactyly type B, bilateral brachydactyly of the 2nd (although the 4th and 5th fingers are also affected), and bilateral complete cutaneous webbing of the 4th and 5th fingers (type 3) with camptodactyly. C: The right foot shows postaxial polydactyly type A. Brain magnetic resonance imaging, shows parietal cortical atrophy and hypoplasia (arrowheads) of both (D) the olivopontocerebellar and (H) corpus callosum. X-rays show (E) scoliosis, (F) brachydactyly of the middle phalanx of the 2nd, 4th, and 5th fingers, bilateral bony syndactyly of the 4th and 5th fingers, bilateral postaxial polydactyly type-B in the hands, and (G) PAP-A in the right foot with duplication of the distal phalanx of the left 4th toe.

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Brain magnetic resonance imaging showed parietal cortical atrophy and hypoplasia of both the olivopontocerebellar and corpus callosum (Fig. 3D,H) and renal ultrasonography showed hypoplastic kidneys (8.44 cm, data not shown). X-rays showed scoliosis (Fig. 3E), bilateral bony syndactyly and brachydactyly (Fig. 3F) as well as PAP-A in the right foot with a duplication of the distal phalanx of the left 4th toe (Fig. 3G).

Patient IV.17 (45 years old) presented with a phenotype almost identical to that of the index case (Fig. 2D–F) except for the lack of hand polydactyly. This patient had vertical creases in his forehead and left divergent strabismus. Patient V.12 (28 years old) had milder intellectual disability, but the dysmorphic facial features were virtually the same (Fig. 2G) except for the absence of ptosis and low-set ears. The limb abnormalities displayed slight variations, such as absence of the bilateral duplication of the distal phalanx of the left 4th toe, incomplete syndactyly in the hands and a bifid nail in the 3rd left toe (Fig. 2H,I). Patient V.17 (24 years old) had the mildest intellectual disability; he has developed appropriate social and communication skills and completed elementary education. With some parental supervision, he can attend to his own personal care. This patient's phenotype (Fig. 2J) was more similar to that of V.12. In particular, this patient had mandibular prognathism, left foot polydactyly, and bilateral clinodactyly and brachydactyly with a total absence of the distal interphalangeal creases in the 2nd, 4th, and 5th fingers (Fig. 2K,L).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CLINICAL REPORT
  5. MATERIALS AND METHODS
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Genetic Studies

Blood samples were collected by venipuncture after the patients' guardians signed the informed consent forms. The chromosome analyses of the index case and his parents were performed according to standard procedures using GTG-banded chromosomes from peripheral blood lymphocytes [Brown and Lawce, 1997].

Molecular Analyses

A 105 K microarray-based comparative genome hybridization (aCGH) that surveys >150 clinically significant chromosomal loci, gene-dense regions, all subtelomeres, the pericentromeric regions and the entire X chromosome was used to analyze the index case and his parents (GeneDx; Gaithersburg, MD). The X chromosome was 2–10 times more densely covered by probes.

Linkage Analysis

Genomic DNA was isolated from blood samples from the four mother–son pairs and IV.21, or from buccal swab samples from other family members (II.4, IV.18) using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). The X chromosome was genotyped using the ABI Linkage Mapping Set HD5 (Applied Biosystems, Foster City, CA), which detects 48 microsatellite markers at a resolution of approximately 5 cM. The amplified products were resolved using an ABI PRISM 3100 Genetic Analyzer and examined with the GeneMapper software (Applied Biosystems). The chromosomal phases were manually obtained by haplotype analysis. A parametric two-point genetic linkage analysis was manually performed using the original formula [Morton, 1955]. Complete penetrance in males, mutation like allele frequency and a lack of locus heterogeneity were assumed for this analysis. To maximize the genetic linkage, the final LOD score was evaluated as the sum of four independent genetic linkages of which each analysis included the four patients (one at a time) along with the four mothers.

X Chromosome Inactivation (XCI) Analysis

We performed the human androgen receptor (HUMARA) X-inactivation assay [Allen et al., 1992] using modifications for fluorescence detection. Briefly, 6-FAM™ PCR products were resolved and analyzed as in the genotyping procedure. The XCI percentages were calculated as described by Sharp et al. [2000].

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CLINICAL REPORT
  5. MATERIALS AND METHODS
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Molecular Analyses

Neither chromosomal alterations nor copy number variations were found using 550 GTG-banding karyotypes or aCGH, respectively (data not shown).

Linkage Analysis

In order to map the phenotype, we conducted a linkage analysis that focused on the X chromosome based on pedigree analysis. The linkage analysis results showed one locus on the short arm and two loci on the long arm of the X chromosome, all these with significant LOD scores of theta (θ) = 0.0. The first locus located on the short arm of the X chromosome was composed of the four markers DXS993–DXS1055, which have LOD scores of 4.8, and the two markers DXS1039 and DXS991, which have LOD scores of 3.6 (Fig. 1). The second locus included an interval between markers DXS1196 and DXS1217 with LOD score values of 4.8 and 2.4, respectively (Fig. 1). The third locus was an isolated marker, DXS8088 that had a LOD score of 3.6 (data not shown). Haplotype inspection revealed the first locus as a candidate region that mapped to Xp11.4–p11.21 and covered 15.74 Mb. This genomic region was located between the limiting markers DXS8015 and DXS991. At the second candidate locus, the mother III.7 resembled to be homozygous for the haplotype linked to the phenotype. At least three different haplotypes were implied at the third locus, and mothers IV.7 and IV.10 were both homozygous for some of them.

As expected for a healthy male, individual IV.21 inherited a haplotype from his mother (III.7) other than the one that was found associated with the disease phenotype, while his sister (IV.18) was found to be heterozygous for the disease haplotype at the candidate region (Fig. 1).

Differential Diagnosis

To determine whether this disease is indeed the result of an allelic disorder, we performed database (PubMed, http://www.ncbi.nlm.nih.gov/pubmed, POSSUM, http://www.possum.net.au/, and Genatlas) analyses. Bioinformatics tools predicted the presence of an allelic form of four possible multiple congenital anomaly syndromes. One of these syndromes, the Simpson–Golabi–Behmel syndrome [Golabi et al., 1993] (SGBS, Xq26.2, OMIM:312870), is comparatively similar and exhibits similar polydactyly, brachydactyly, syndactyly, intellectual disability, scoliosis, and inheritance patterns. However, SGBS is an overgrowth syndrome that is inconsistent with the short stature, brachycephaly and hypoplastic kidneys that were described in this family. Greig cephalopolysyndactyly syndrome [Biesecker, 2008] (GCPS, 7p14.1, OMIM:175700) is another overgrowth syndrome that presents with polydactyly and intellectual disability but differs in the types of polydactyly (preaxial in feet), and syndactyly (3rd and 4th fingers, and 1st–3rd toes). X-linked oral–facial–digital syndrome type I [Toriello and Franco, 1993; Feather et al., 1997] (OFD1, Xp22.2, OMIM:311200) shares the polysyndactyly, brachydactyly, and intellectual disability phenotypes, but features such as hamartomas, polycystic kidneys and lethality in males are exclusive to OFD1. Finally, the autosomal recessive oral–facial–digital syndrome type II [Toriello and Franco, 1993] (OFD2; OMIM:252100) only phenotypically overlaps with the brachydactyly and postaxial polydactyly symptoms.

Bioinformatic Analysis

According to the Ensembl and Genatlas databases, the Xp11.4–p11.21 region contains a total of 250 genes, of which 145 are confirmed protein-encoding genes and another 23 are thought to be protein-encoding genes. In addition, only 63 of these genes (43%) are reported by the OMIM database as being associated with known phenotypes. Finally, a search for phenotypes of the genes in the candidate region which are not associated with known syndromes in model animals was performed using both the mouse genome informatics (http://www.informatics.jax.org/) and the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/mouseportal/) databases. Phenotypes were only reported for the (MOUSE) Timp1, Phf16, and Med14 genes; these models were not relevant to our study.

X-Chromosome Inactivation (XCI) Analysis

We found that mothers III.7 and IV.10 possessed skewed XCI patterns (80:20) for the X chromosomes harboring the alteration, whereas mothers III.3 and IV.7 showed random patterns of XCI (48:52 and 46:54, respectively; Fig. 4). Three out of four mothers (III.3, III.7, and IV.7) had the same androgen receptor (AR) allele (276 bp) as their respective sons (IV.5, IV.17, and V.12) within the haplotype linked to the disease phenotype (asterisk in Figs. 1 and 4). However, in mother IV.10, a previous recombination event had linked a different AR allele (279 bp) to the mutant haplotype that was inherited by V.17 (Figs. 1 and 4).

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Figure 4. Two different patterns of X-chromosome inactivation found between the obligate carrier mothers. Electropherograms show the PCR products that were derived from the AR gene amplification using genomic DNA, which was either native (upper tracks) or previously digested with the HpaII restriction enzyme (lower tracks). The pedigree location for each mother is shown at the top. The percentage of inactivation for each AR allele is given below the electropherograms, which show a skewed pattern for mothers III.7 and IV.10 and a random pattern for mothers III.3 and IV.7. The AR allele co-segregating with the involved haplotype is represented in bold. The size of each expected AR allele is indicated by both blue bins and gray bars above the electropherograms. [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/ajmga]

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CLINICAL REPORT
  5. MATERIALS AND METHODS
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

In this report, we describe a Mexican family with a novel intellectual disability syndrome thought to be inherited as an X-linked recessive disorder. The phenotype does not match with known syndromes, which suggests that this likely is a novel genetic entity. This condition is characterized by facial anomalies, digital abnormalities, and short stature as the major features. Moreover, we considered characteristics such as microbrachycephaly, scoliosis, hypoplasia and atrophy of some organs as minor criteria. The digital abnormalities could be the signature characteristic of this phenotype; specifically bilateral PAP-B, a unique bilateral brachydactyly of the 2nd/4th/5th fingers (4th finger is absent in brachydactyly type A), bilateral camptodactyly and bilateral cutaneous webbing of the 4th/5th fingers. Digital anomalies of the feet included bilateral cutaneous webbing of the 2nd/3rd toes and right or left PAP-A.

We also observed a variable intrafamilial expression of the disorder; for example, the skeletal abnormalities and intellectual disability were more severe in patients from generation IV compared with V.

The identification of the locus Xp11.4–p11.21 by linkage and haplotype analyses confirmed the X-linked mode of inheritance of this phenotype and supports the idea of this as a novel genetic entity. Interestingly, the Patient V.17, who was the most informative in the linkage analysis from having inherited the shortest haplotype, is also the least affected of the four patients. Conversely, Patients IV.5 and IV.17, who harbor the largest haplotypes, were the more affected patients. This appears to suggest that genetic factors other than the mutation, either in the same genomic region or more likely in the genetic background, can modify the expressivity of this disorder between both generations to some extent.

Because this X-linked recessive phenotype does not show a progressive course, we speculate that the expression of the involved gene is temporally and spatially regulated during embryonic development. Moreover, the constellation of symptoms likely reflects the expression pattern of the gene, the locations of its activity, or both. The gene involved in this phenotype likely encodes a regulatory protein with functions similar to those that are responsible for other multiple congenital anomaly syndromes in which cellular processes such as proliferation and apoptosis are impaired.

Since neither chromosomal alterations nor copy number variations were found in the index patient, we hypothesize that the alteration is likely a point mutation. Moreover, an amorphic or hypomorphic mutation should be involved; otherwise, at least heterozygous mothers harboring random XCI patterns would express the disease [Dobyns et al., 2004], which did not happen.

Assuming that this phenotype is a novel and specific monogenic syndrome, the candidate gene in this region must be among the 28 protein-encoding genes reported in Genatlas database which are not associated with a phenotype or which have unknown function. Candidate genes ZNF182, WDR13, and WDR45 encode regulatory molecules which play roles in proliferation, differentiation and apoptosis. Genes such as ZNF182, PRICKLE3, and ZNF157 encode transcription factors, while PHF16 gene encodes a component of the histone H4 acetyltransferase complex. Specifically, the helicase-encoding gene DDX3X and the transcription factor-encoding gene TSPYL2 have Y chromosome homologs. The genes TIMP1 and CDK16 encoding enzymes, and the regulatory genes MED14 and SUV39H1 are XCI escape genes. Finally, the six genes CXorf38, FUNDC1, CCDC22, FAM156B, FAM156A, and FAM104B remain with unknown function.

A next generation DNA sequencing (NGS) approach or a systematic search for mutations in codifying regions and the intron–exon boundaries of all of the candidate genes will be required to identify the causal mutation of this phenotype. This identification undoubtedly will provide insights into the role of the gene in subtle processes such as controlling the number, growth and individualization of the digits during development as well as its role whose alteration provokes intellectual and motor disabilities, among other features.

In summary, our pedigree analysis, clinical findings, and the genetic linkage and XCI assay results suggest a novel X-linked syndrome in this family.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CLINICAL REPORT
  5. MATERIALS AND METHODS
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

We thank the families for their generous cooperation in this study. We appreciate the help of Viviana Gomez-Puente and Luz Rojas-Patlán in the interpretation of the karyotype studies. This study was conducted using resources from the Departamento de Genética. M.C.B-C. received a fellowship from the Consejo Nacional de Ciencia y Tecnología (CONACyT), México.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CLINICAL REPORT
  5. MATERIALS AND METHODS
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES
  • Allen RC, Zoghbi HY, Moseley AB, Rosenblatt HM, Belmont JW. 1992. Methylation of Hpall and Hhal sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation. Am J Hum Genet 51:12291239.
  • Biesecker LG. 2008. The Greig cephalopolysyndactyly syndrome. Orphanet J Rare Dis 24:310.
  • Brown MG, Lawce J. 1997. Peripheral blood cytogenetic methods. In: Barch J, Knutsen T, Spurbeck J, editors. The AGT cytogenetics laboratory manual, 3rd edition. Philadelphia, PA: Lippincott-Raven. pp 77171.
  • Dobyns WB, Filauro A, Tomson BN, Chan AS, Ho AW, Ting NT, Oosterwijk JC, Ober C. 2004. Inheritance of most X-linked traits is not dominant or recessive, just X-linked. Am J Med Genet Part A 129A:136143.
  • Feather SA, Woolf AS, Donnai D, Malcolm S, Winter RM. 1997. The oral–facial–digital syndrome type 1 (OFD1), a cause of polycystic kidney disease and associated malformations, maps to Xp22.2–Xp22.3. Hum Mol Genet 6:11631167.
  • Golabi M, Leung A, Lopez C. 1993. Simpson–Golabi–Behmel syndrome type 1. 2006 Dec 19 (updated 2011 Jun 23). In: Pagon RA, Bird TD, Dolan CR, Stephens K, Adam MR, editors. GeneReviews™ (Internet). Seattle, WA: University of Washington. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1219/
  • Morton NE. 1955. Sequential tests for the detection of linkage. Am J Hum Genet 7:277318.
  • Sharp A, Robinson D, Jacobs P. 2000. Age- and tissue-specific variation of X chromosome inactivation ratios in normal women. Hum Genet 107:343349.
  • Toriello HV, Franco B. 1993. Oral–facial–digital syndrome type I. 2002 Jul 24 (updated 2010 Oct 14). In: Pagon RA, Bird TD, Dolan CR, Stephens K, Adam MR, editors. GeneReviews™ (Internet). Seattle, WA: University of Washington. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1188/