WNT10B variants in split hand/foot malformation: Report of three novel families and review of the literature

Split‐hand/foot malformation (SHFM) is a genetically heterogeneous congenital limb malformation typically limited to a defect of the central rays of the autopod, presenting as a median cleft of hands and feet. It can be associated with long bone deficiency or included in more complex syndromes. Among the numerous genetic causes, WNT10B homozygous variants have been recently identified in consanguineous families, but remain still rarely described (SHFM6; MIM225300). We report on three novel SHFM families harboring WNT10B variants and review the literature, allowing us to highlight some clinical findings. The feet are more severely affected than the hands and there is a frequent asymmetry without obvious side‐bias. Syndactyly of third–fourth fingers was a frequent finding (62%). Polydactyly, which was classically described in SHFM6, was only present in 27% of patients. No genotype–phenotype correlation is delineated but heterozygous individuals might have mild features of SHFM, suggesting a dose‐effect of the WNT10B loss‐of‐function.     

Split hand/split foot anomaly in a family segregating a balanced translocation with breakpoint on 7q22.1

An apparently balanced translocation, t(2;7)(q21.1;q22.1) was detected in a female patient with bilateral split hand and right split foot. Split hand/split foot (SHSF) segregated as an autosomal dominant character with low penetrance in her family. The translocation was present in 6 of 13 additional relatives investigated, one of whom also had split hand on right. This observation provides further confirmation of the presence of a locus for SHSF on 7q and narrows the critical region to band 7q22.1. Defects caused by alterations of this chromosome region are variable and include manifestations of both syndromal and non-syndromal SHSF. Review of SHSF cases associated with chromosome 7 abnormalities showed a preferential involvement of the lower limbs and of the right side, suggesting the action of locally restricted developmental resistance mechanisms. © 1993 Wiley-Liss, Inc.     

Split hand/foot malformation with long‐bone deficiency and BHLHA9 duplication: report of 13 new families

Split hand/foot malformation (SHFM) with long‐bone deficiency (SHFLD, MIM#119100) is a rare condition characterized by SHFM associated with long‐bone malformation usually involving the tibia. Previous published data reported several unrelated patients with 17p13.3 duplication and SHFLD. Recently, the minimal critical region had been reduced, suggesting that BHLHA9 copy number gains are associated with this limb defect. Here, we report on 13 new families presenting with ectrodactyly and harboring a BHLHA9 duplication.     

少指缺掌并足裂畸形一家系调查及其病因研究

目的　研究先天性少指缺掌并足裂畸形一家系患者的临床表现及其致病原因。方法　在患者家系调查的基础上 ,设立正常对照组与患者组 ,应用聚合酶链反应技术对两组的 P6 3基因第 5～ 8外显子基因组 DNA测序分析。结果　体检发现 ,患者双上肢少拇指、食指和中指 ,缺桡侧手掌并双下肢足裂畸形。病因研究提示 ,P6 3基因第 5～ 8外显子 PCR扩增片段大小分别为 2 84 bp、2 5 9bp、2 4 5 bp和 2 5 9bp。患者与正常对照的扩增片段大小一致。但将患者扩增的片段 DNA序列分别与正常对照组以及人类基因库P6 3基因 DNA序列进行比较时则发现于 P6 3基因第 5外显子的第 6 6 5位碱基对出现突变 ,即由 G突变为A。结论　先天性少指缺掌并足裂家系患者系由 P6 3基因第 5外显子基因组的第 6 6 5位碱基对突变所致。     

Characterization of a complex rearrangement of a chromosome 20 by FISH and array CGH

We report on a patient with a Complex Chromosomal Rearrangement (CCR) of a chromosome 20. CCRs are structural rearrangements involving three or more chromosomes or having more than two breakpoints; congenital CCRs compatible with life are a rare finding in humans. The rearranged chromosome 20 was characterized by array Comparative Genomic Hybridization (array CGH), and Fluorescent In Situ Hybridization (FISH). The combination of these two techniques made it possible to precisely define the rearrangement and showed a very unusual architecture, with segments deleted, duplicated, inverted, and shifted. Potential mechanisms generating such a complex rearrangement and candidate genes responsible for the phenotype are discussed.     

一个中国人裂手足伴并指趾畸形家系的表型和基因型分析

目的 确定一个中国人裂手足家系的致病性基因突变,探讨基因型-表型关系.方法 收集患者及其家庭成员的外周血,提取基因组DNA.采用PCR扩增P63基因和HOXD13基因的外显子,对PCR产物进行双向测序检测基因突变.结果 在所有患者中检测到P63基因第7外显子的杂合型956G→A突变,导致蛋白产物第280位的精氨酸被组氨酸取代(R280H).在正常家庭成员中未检测到该突变.结论 该家族中的患者以对称性裂手足伴并指趾为主要特征,由P63基因的R280H突变所致.     

Disclosure of five breakpoints in a complex chromosome rearrangement by microdissection and FISH.

Microdissection and fluorescence in situ hybridisation (FISH) were used to elucidate the nature of a complex chromosome translocation, after GTG banding failed in the complete characterisation of the structural rearrangement between chromosomes 6 and 12. These chromosomes were painted with chromosome specific paints and one of the chromosome regions involved in the translocation was isolated by microdissection. Ten copies of the microdissected region were collected with microneedles from GTG banded metaphases, transferred to a collecting drop, and amplified by means of DOP-PCR. The PCR product was labelled with biotin-14-dATP and used as a FISH probe for hybridisation to normal metaphase chromosomes and metaphase chromosomes of the patients (microFISH). FISH with this chromosome region specific painting probe and with chromosome band specific probes enabled the characterisation of a complex chromosome rearrangement with five breakpoints in two chromosomes. This resulted in the following karyotype: 46,XY,t(6;12)(6pter--> 6q12::12q24.1-->12qter;12qter-->12q13.3:: 6q16.2-->6q26::12q13.3-->12q24.1::6q12--> 6q16.2::6q26-->6qter).     

TP63 mutation and clefting modifier genes in an EEC syndrome family

Autosomal dominant EEC syndrome consists of ectrodactyly, ectodermal dysplasia, and cleft lip with or without cleft palate (CL/P). We investigated an EEC kindred with 10 affected persons in three generations in order to map the causative mutation in this family and to map modifier genes that contribute to the expression of facial clefting in the phenotype. DNA from 15 family members was genotyped for 388 genome screen markers. Analysis revealed maximal linkage between EEC and chromosome 3q27, which contains a known EEC gene - tumor protein 63 (TP63). Sequencing showed a CGT-->TGT missense mutation (R280C) in exon 7, previously reported to cause EEC in four families, and ectrodactyly alone (split hand-foot malformation) in one sporadic case and one large kindred. Analysis of the clefting phenotype in this EEC family demonstrated maximal linkage to two regions on chromosomes 4q and 14, which multiple studies have implicated in non-syndromic CL/P. In conclusion, this study demonstrates that the mutation of TP63 is the major (Mendelian) EEC gene in this kindred and suggests that additional minor modifying genes which predispose to non-syndromic CL/P could also contribute to the expression of the clefting component of the syndrome in this family.     

Comprehensive characterization of genomic aberrations in gangliogliomas by CGH, array-based CGH and interphase FISH

Gangliogliomas are generally benign neuroepithelial tumors composed of dysplastic neuronal and neoplastic glial elements. We screened 61 gangliogliomas [World Health Organization (WHO) grade I] for genomic alterations by chromosomal and array-based comparative genomic hybridization (CGH). Aberrations were detected in 66% of gangliogliomas (mean ± SEM = 2.5 ± 0.5 alterations/tumor). Frequent gains were on chromosomes 7 (21%), 5 (16%), 8 (13%), 12 (12%); frequent losses on 22q (16%), 9 (10%), 10 (8%). Recurrent partial imbalances comprised the minimal overlapping regions dim(10)(q25) and enh(12)(q13.3–q14.1). Unsupervised cluster analysis of genomic profiles detected two major subgroups (group I: complete gain of 7 and additional gains of 5, 8 or 12; group II: no major recurring imbalances, mainly losses). A comparison with low-grade gliomas (astrocytomas WHO grade II) showed chromosome 5 gain to be significantly more frequent in gangliogliomas. Interphase fluorescence in situ hybridization (FISH) identified the aberrations to be contained in a subpopulation of glial but not in neuronal cells. Two gangliogliomas and their anaplastic recurrences (WHO grade III) were analyzed. Losses of CDKN2A/B and DMBT1 or a gain/amplification of CDK4 found in the anaplastic tumors were already present in the respective gangliogliomas by array CGH and interphase FISH. In summary, genomic profiling in a large series of gangliogliomas could distinguish genetic subgroups even in this low-grade tumor.     

Characterization of a complex chromosome rearrangement involving 6q in a melanoma cell line by chromosome microdissection

Deletion of 6q is one of the most frequent chromosomal alterations in human malignant melanoma. Recently, we used chromosome painting probes of 6p and 6q to study 21 melanoma cell lines. A reciprocal translocation between chromosomes 6q and 17p was detected in one cell line (UACC-930). Upon further characterization of the translocation marker using the micro fluorescence in situ hybridization (FISH) technique, a complex rearrangement including an inversion of 6q and a translocation between the inverted 6q and 17p, [der(6)inv(6)(q16q27)t(6;17)(q26;p13)], was detected. A yeast artificial chromosome (YAC) clone spanning the breakpoint at 6q16 was isolated by the FISH screen. Loss of one or more copies of the YAC clone was also detected in 10 of 12 melanoma cell lines. This result implies that the YAC clone may contain a putative tumor suppressor gene related to the pathogenesis of malignant melanoma. Further characterizations of the breakpoint at 6q16 and molecular cloning breakpoints at 6q27 and 17p13 are in progress.     

Split hand/split foot malformation, deafness, and mental retardation with a complex cytogenetic rearrangement involving 7q21.3.

Split hand/split foot malformation (SHSF) has been described in several patients associated with cytogenetically visible rearrangements involving chromosome 7q. Characterisation of these patients has led to localisation of an autosomal dominant form of SHSF to 7q21-22; the locus has been designated SHFM1. We describe a patient with a complex, apparently balanced cytogenetic rearrangement, including a translocation breakpoint at 7q21.3 near the DSS1 gene. In addition to ectrodactyly of all four limbs, the patient has congenital deafness, submucous cleft palate, microcephaly, and mental retardation. This patient represents an additional case of syndromic ectrodactyly related to the SHFM1 gene region, which may be responsible for both syndromic and non-syndromic ectrodactyly.     

Cytogenetic and array CGH characterization of an intrachromosomal complex rearrangement of 4q in a patient with a 4q-phenotype

We report on a 3-year-old child who presented a de novo rearrangement of chromosome 4, detected on GTG banding and characterized by array CGH and FISH, as a complex intrachromosomal rearrangement with three deletions: del(q32.1q32.2), del(q33q34.1), del(q35.2), one tandem duplication dup(q34.3q35.1) and short normal regions in between. The study of karyotype-phenotype correlations in this and other patients with deletions of 4q suggests 4q33q34.1 as a candidate region for 4q-syndrome and for craniofacial development.     

Epilepsy with cognitive deficit and autism spectrum disorders: Prospective diagnosis by array CGH

The clinical significance of chromosomal microdeletions and microduplications was predicted based on their gene content, de novo or familial inheritance and accumulated knowledge recorded on public databases. A patient group comprised of 247 cases with epilepsy and its common co‐morbidities of developmental delay, intellectual disability, autism spectrum disorders, and congenital abnormalities was reviewed prospectively in a diagnostic setting using a standardized oligo‐array CGH platform. Seventy‐three (29.6%) had copy number variations (CNVs) and of these 73 cases, 27 (37.0%) had CNVs that were likely causative. These 27 cases comprised 10.9% of the 247 cases reviewed. The range of pathogenic CNVs associated with seizures was consistent with the existence of many genetic determinants for epilepsy. © 2012 Wiley Periodicals, Inc.     

Directly inherited partial trisomy of chromosome 6p identified in a father and daughter by chromosome microdissection

Cytogenetic analysis of a 4 year old girl with developmental delay and dysmorphic features showed extra chromosomal material of unknown origin on 20p (46,XX,add(20)(p13)). Familial chromosome studies showed direct inheritance of add(20)(p13) from the father, who had a similar, albeit milder, phenotype. Fibroblast chromosome studies of the father showed no karyotype mosaicism. The additional material could not be identified on the basis of the G banding pattern owing to its small size and ambiguous banding pattern. Chromosome microdissection of the unknown material was performed, the DNA was amplified and labelled using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and reverse painted to the proband's cells to show the karyotype 46,XX,der(20)t(6;20) (p23;p13), conferring partial trisomy 6p and presumed partial monosomy for 20p. Chromosome microdissection has made possible the first reported case of directly inherited partial trisomy 6p.     

Identification and analysis of sex chromosomes by comparative genomic hybridization (CGH)

Comparative Genome Hybridization (CGH) can be used as a universal method for the identification of molecularly differentiated sex chromosomes. This is profitable in species with homomorphic sex chromosomes or when chromosomes are unfavourable for cytogenetics, e.g. when size differences are insufficient, chromosomes numerous and/or banding methods fail. In this method, genomic DNA from females competes as a probe with that from males for binding to the chromosome targets. Easy extraction and labelling methods afford a method that can be applied even when few specimens are available, e.g. when specimens for investigation have to be collected in the field – CGH also offers the possibility to obtain a rough estimate of the DNA composition of the sex chromosome.     

TWIST microdeletion identified by array CGH in a patient presenting Saethre-Chotzen phenotype and a complex rearrangement involving chromosomes 2 and 7

Saethre-Chotzen syndrome (SCS), also known as acrocephalosyndactyly III, is an autosomal dominant hereditary disorder characterized by craniofacial and limb anomalies. SCS is generally caused by mutations in the TWIST gene, but several 7p21.3 microdeletions involving the entire gene have also been described. The patient reported here presented with craniosynostosis, ptosis, brachydactyly and syndactyly of toes. Standard lymphocyte karyotype showed a de novo apparently balanced but complex constitution with a translocation between the short arms of chromosomes 2 and 7 and an insertion of the 7(q21.3q22) band in the short arm of the same chromosome 7. Interestingly, array CGH displayed a unique 690 kb deletion in 7p21.3 involving the TWIST gene, consistent with the phenotype. This case illustrates the important contribution of array CGH to identification of complex chromosomal rearrangements.     

Duplication 16q12.1-q22.1 characterized by array CGH in a girl with spina bifida

We report a 7-year-old girl with spina bifida carrying a complex chromosome abnormality resulting in duplication 16q12.1-q22.1. An abnormal karyotype was identified involving the long arm of chromosome 11 and fluorescent in situ hybridization (FISH) to metaphase chromosomes revealed an insertion of part of chromosome 16 on chromosome 11. A detailed mapping of the chromosome abnormality using whole genome array based comparative genomic hybridization (CGH) of the patient DNA revealed a duplication 16q12.1-q22.1 corresponding to gain of 19.8Mb of DNA without any detectable loss of genetic material on chromosome 11. The karyotype is defined as 46,XX,der(11)ins(11;16)(q13;q12.1q22.1). We present here the clinical findings and a fine mapping of the associated structural chromosome abnormalities. We suggest that a gene dosage imbalance of 16q12.1-q22.1 is associated with spina bifida in the patient.