Aleksander Jamsheer

Duplications of BHLHA9 are associated with ectrodactyly and tibia hemimelia inherited in non-Mendelian fashion

Background Split-hand/foot malformation (SHFM)—also known as ectrodactyly—is a congenital disorder characterised by severe malformations of the distal limbs affecting the central rays of hands and/or feet. A distinct entity termed SHFLD presents with SHFM and long bone deficiency. Mouse models suggest that a defect of the central apical ectodermal ridge leads to the phenotype. Although six different loci/mutations (SHFM1–6) have been associated with SHFM, the underlying cause in a large number of cases is still unresolved. Methods High resolution array comparative genomic hybridisation (CGH) was performed in patients with SHFLD to detect copy number changes. Candidate genes were further evaluated for expression and function during limb development by whole mount in situ hybridisation and morpholino knock-down experiments. Results Array CGH showed microduplications on chromosome 17p13.3, a locus previously associated with SHFLD. Detailed analysis of 17 families revealed that this copy number variation serves as a susceptibility factor for a highly variable phenotype with reduced penetrance, particularly in females. Compared to other known causes for SHFLD 17p duplications appear to be the most frequent cause of SHFLD. A ∼11.8 kb minimal critical region was identified encompassing a single gene, BHLHA9, a putative basic loop helix transcription factor. Whole mount in situ hybridisation showed expression restricted to the limb bud mesenchyme underlying the apical ectodermal ridge in mouse and zebrafish embryos. Knock down of bhlha9 in zebrafish resulted in shortening of the pectoral fins. Conclusions Genomic duplications encompassing BHLHA9 are associated with SHFLD and non-Mendelian inheritance characterised by a high degree of non-penetrance with sex bias. Knock-down of bhlha9 in zebrafish causes severe reduction defects of the pectoral fin, indicating a role for this gene in limb development.

A novel nonsense mutation in CUL4B gene in three brothers with X-linked mental retardation syndrome

Badura‐Stronka M, Jamsheer A, Materna‐Kiryluk A, Sowińska A, Kiryluk K, Budny B, Latos‐Bieleńska A. A novel nonsense mutation in CUL4B gene in three brothers with X‐linked mental retardation syndrome.

Phenotype and genotype in 103 patients with tricho-rhino-phalangeal syndrome.

Tricho-rhino-phalangeal syndrome (TRPS) is characterized by craniofacial and skeletal abnormalities, and subdivided in TRPS I, caused by mutations in TRPS1, and TRPS II, caused by a contiguous gene deletion affecting (amongst others) TRPS1 and EXT1. We performed a collaborative international study to delineate phenotype, natural history, variability, and genotype-phenotype correlations in more detail. We gathered information on 103 cytogenetically or molecularly confirmed affected individuals. TRPS I was present in 85 individuals (22 missense mutations, 62 other mutations), TRPS II in 14, and in 5 it remained uncertain whether TRPS1 was partially or completely deleted. Main features defining the facial phenotype include fine and sparse hair, thick and broad eyebrows, especially the medial portion, a broad nasal ridge and tip, underdeveloped nasal alae, and a broad columella. The facial manifestations in patients with TRPS I and TRPS II do not show a significant difference. In the limbs the main findings are short hands and feet, hypermobility, and a tendency for isolated metacarpals and metatarsals to be shortened. Nails of fingers and toes are typically thin and dystrophic. The radiological hallmark are the cone-shaped epiphyses and in TRPS II multiple exostoses. Osteopenia is common in both, as is reduced linear growth, both prenatally and postnatally. Variability for all findings, also within a single family, can be marked. Morbidity mostly concerns joint problems, manifesting in increased or decreased mobility, pain and in a minority an increased fracture rate. The hips can be markedly affected at a (very) young age. Intellectual disability is uncommon in TRPS I and, if present, usually mild. In TRPS II intellectual disability is present in most but not all, and again typically mild to moderate in severity. Missense mutations are located exclusively in exon 6 and 7 of TRPS1. Other mutations are located anywhere in exons 4-7. Whole gene deletions are common but have variable breakpoints. Most of the phenotype in patients with TRPS II is explained by the deletion of TRPS1 and EXT1, but haploinsufficiency of RAD21 is also likely to contribute. Genotype-phenotype studies showed that mutations located in exon 6 may have somewhat more pronounced facial characteristics and more marked shortening of hands and feet compared to mutations located elsewhere in TRPS1, but numbers are too small to allow firm conclusions.

Osteopoikilosis and multiple exostoses caused by novel mutations in LEMD3 and EXT1 genes respectively - coincidence within one family

BackgroundOsteopoikilosis is a rare autosomal dominant genetic disorder, characterised by the occurrence of the hyperostotic spots preferentially localized in the epiphyses and metaphyses of the long bones, and in the carpal and tarsal bones [1]. Heterozygous LEMD3 gene mutations were shown to be the primary cause of the disease [2]. Association of the primarily asymptomatic osteopokilosis with connective tissue nevi of the skin is categorized as Buschke-Ollendorff syndrome (BOS) [3]. Additionally, osteopoikilosis can coincide with melorheostosis (MRO), a more severe bone disease characterised by the ectopic bone formation on the periosteal and endosteal surface of the long bones [4–6]. However, not all MRO affected individuals carry germ-line LEMD3 mutations [7]. Thus, the genetic cause of MRO remains unknown. Here we describe a familial case of osteopoikilosis in which a novel heterozygous LEMD3 mutation coincides with a novel mutation in EXT1, a gene involved in aetiology of multiple exostosis syndrome. The patients affected with both LEMD3 and EXT1 gene mutations displayed typical features of the osteopoikilosis. There were no additional skeletal manifestations detected however, various non-skeletal pathologies coincided in this group.MethodsWe investigated LEMD3 and EXT1 in the three-generation family from Poland, with 5 patients affected with osteopoikilosis and one child affected with multiple exostoses.ResultsWe found a novel c.2203C > T (p.R735X) mutation in exon 9 of LEMD3, resulting in a premature stop codon at amino acid position 735. The mutation co-segregates with the osteopoikilosis phenotype and was not found in 200 ethnically matched controls. Another new substitution G > A was found in EXT1 gene at position 1732 (cDNA) in Exon 9 (p.A578T) in three out of five osteopoikilosis affected family members. Evolutionary conservation of the affected amino acid suggested possible functional relevance, however no additional skeletal manifestations were observed other then those specific for osteopoikilosis. Finally in one member of the family we found a splice site mutation in the EXT1 gene intron 5 (IVS5-2 A > G) resulting in the deletion of 9 bp of cDNA encoding three evolutionarily conserved amino acid residues. This child patient suffered from a severe form of exostoses, thus a causal relationship can be postulated.ConclusionsWe identified a new mutation in LEMD3 gene, accounting for the familial case of osteopoikilosis. In the same family we identified two novel EXT1 gene mutations. One of them A598T co-incided with the LEMD3 mutation. Co-incidence of LEMD3 and EXT1 gene mutations was not associated with a more severe skeletal phenotype in those patients.

Whole exome sequencing identifies FGF16 nonsense mutations as the cause of X-linked recessive metacarpal 4/5 fusion

Background Metacarpal 4–5 fusion (MF4; MIM %309630) is a rare congenital malformation of the hand characterised by the partial or complete fusion of the fourth and fifth metacarpals. The anomaly occurs as an isolated trait or part of a genetic syndrome. Methods To search for disease-causing mutation, whole exome sequencing (WES) was performed on samples from a single trio. Before WES, molecular screening including gene sequencing and array comparative genomic hybridisation was applied. Validation of WES and segregation studies were done using routine Sanger sequencing. Results Exome sequencing detected a nonsense mutation (c.C535T; p.R179X) in exon 3 of the FGF16 gene, which maps to chromosome Xq21.1. Mutational screening of the FGF16 gene performed in an unrelated proband of different ethnicity showed another nonsense mutation in exon 3 (c.C470A; p.S157X). Conclusions This study shows that truncating mutations of FGF16 are associated with X-linked recessive metacarpal 4–5 fusion. The study provides evidence for the involvement of FGF16 in the fine tuning of the human skeleton of the hand.

PAX6 3' deletion in a family with aniridia.

Background: Aniridia is a congenital panocular malformation defined as iris aplasia or hypoplasia. It can be either isolated or be a part of multiple ocular anomalies such as cataracts, glaucoma, corneal pannus, optic nerve hypoplasia, absence of macular reflex or ectopia lentis. In the majority of cases the disease is caused by mutation in the PAX6 gene. Material and Methods: A Polish family with aniridia was screened for the presence of genomic rearrangements in PAX6, WT1 and the flanking genes by means of multiplex ligation probe amplification (MLPA). MLPA reaction was performed using the P219-B1 PAX6 commercial kit from MRC-Holland. Additionally, the coding sequence of PAX6 gene was sequenced in the proband. Array comparative genomic hybridization analysis was performed using the NimbleGen CGX-12 format. Results: MLPA examination revealed a heterozygous deletion of approximately 0.6 Mb, downstream of PAX6 gene on chromosome 11. Four genes lie in the deleted region. Bi-directional sequencing of 14 exons of the PAX6 gene did not reveal any causative alteration. Microarray analysis confirmed the deletion and determined its size which ranged from 598.87–651.76 kb. Conclusions: A small subset of aniridia cases is caused by rearrangements of PAX6 neighboring regions, and the so-called “position effect” is considered to be the underlying pathogenic mechanism. Molecular testing of aniridia patients should include sequencing of the PAX6 gene, followed by screening for larger structural abnormalities located on chromosome 11p13. MLPA can be a useful method in molecular testing of aniridia patients.

Isolated brachydactyly type E caused by a HOXD13 nonsense mutation: a case report.

BackgroundBrachydactyly type E (BDE; MIM#113300) is characterized by shortening of the metacarpal, metatarsal, and often phalangeal bones, and predominantly affects postaxial ray(s) of the limb. BDE may occur as an isolated trait or as part of a syndrome. Isolated BDE is rare and in the majority of cases the molecular pathogenesis has so far not been resolved. Originally, the molecular cause of isolated BDE has been unravelled in 2 families and shown to result from heterozygous missense mutations in the homeodomain of the HOXD13 gene. Since the initial manuscript, one further HOXD13 mutation has been reported only in a single family manifesting isolated BDE.Case PresentationIn this paper, we report on a Polish family exhibiting isolated BDE caused by a novel nonsense heterozygous HOXD13 mutation. We investigated a Polish female proband and her father, both affected by isolated BDE, in whom we identified a nonsense heterozygous mutation c.820C > T(p.R274X) in the HOXD13 gene. So far, only two missense HOXD13 substitutions (p.S308C and p.I314L), localized within the homeodomain of the HOXD13 transcription factor, as well as a single nonsense mutation (p.E181X) were associated with BDE. Both missense changes were supposed to alter DNA binding affinity of the protein.ConclusionThe variant p.R274X identified in our proband is the fourth HOXD13 mutation, and the second truncating (nonsense) mutation, reported to result in typical isolated BDE. We refer our clinical and molecular findings to the previously described HOXD13 associated phenotypes and mutations.

A novel GJA1 missense mutation in a Polish child with oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) (OMIM #164200) is a rare congenital, autosomal dominant disorder comprising craniofacial, ocular, dental, and digital anomalies. The syndrome is caused byGJA1 mutations. The clinical phenotype of ODDD involves a characteristic dysmorphic facies, ocular findings (microphthalmia, microcornea, glaucoma), syndactyly type III of the hands, phalangeal abnormalities, diffuse skeletal dysplasia, enamel dysplasia, and hypotrichosis. In a Polish child with the clinical symptoms typical of ODDD, we demonstrated a novel missense mutation c.C31T resulting in p.L11F substitution. Our report provides evidence on the importance of this highly conserved amino acid residue for the proper functioning of GJA1 protein.

Heterozygous DLX5 nonsense mutation associated with isolated split‐hand/foot malformation with reduced penetrance and variable expressivity in two unrelated families

BACKGROUND Split-hand/foot malformation (SHFM) is a clinically and genetically heterogeneous limb abnormality characterized by the absence or hypoplasia of the central rays of the autopod. SHFM1, which is one out of seven known SHFM loci, maps to 7q21.2-q21.3. SHFM1 is usually inherited as an autosomal dominant trait with reduced penetrance, although recessive inheritance has been described for a single family carrying a homozygous DLX5 missense variant. In most cases, SHFM1 results from heterozygous deletions encompassing DLX5/DLX6 genes or from inversions and translocations separating the genes from their limb specific enhancers. Recently, a single Chinese family with dominant SHFM1 was shown to result from a heterozygous DLX5 missense mutation. METHODS In this study, we report on four male individuals from two unrelated Polish families (one sporadic and one familial case) presenting with isolated SHFM. We tested both probands for known molecular causes of SHFM, including TP63, WNT10B, DLX5 mutations and copy number changes using 1.4 M array CGH. RESULTS Sanger sequencing of DLX5 revealed a novel heterozygous nonsense mutation c.G115T(p.E39X) in both index patients. Segregation studies demonstrated that the variant was present in all affected family members but also in three apparently healthy relatives (two females and one male). CONCLUSION This is the first report of a heterozygous DLX5 nonsense mutation resulting in incompletely penetrant autosomal dominant isolated SHFM1. Data shown here provides further evidence for the contribution of DLX5 point mutations to the development of ectrodactyly and suggest the possibility of sex-related segregation distortion with an excess of affected males.

MIA is a potential biomarker for tumour load in neurofibromatosis type 1

BackgroundNeurofibromatosis type 1 (NF1) is a frequent genetic disease characterized by multiple benign tumours with increased risk for malignancy. There is currently no biomarker for tumour load in NF1 patients.MethodsIn situ hybridization and quantitative real-time polymerase reaction were applied to investigate expression of cartilage-specific genes in mice bearing conditional inactivation of NF1 in the developing limbs. These mice do not develop tumours but recapitulate aspects of NF1 bone dysplasia, including deregulation of cartilage differentiation. It has been recently shown that NF1 tumours require for their growth the master regulator of cartilage differentiation SOX9. We thus hypothesized that some of the cartilage-specific genes deregulated in an Nf1Prx1 mouse model might prove to be relevant biomarkers of NF1 tumours. We tested this hypothesis by analyzing expression of the SOX9 target gene product melanoma-inhibitory activity/cd-rap (MIA) in tumour and serum samples of NF1 patients.ResultsIncreased expression of Mia was found in Nf1-deficient cartilage in mice. In humans, MIA was expressed in all NF1-related tumours and its serum levels were significantly higher in NF1 patients than in healthy controls. Among NF1 patients, MIA serum levels were significantly higher in those with plexiform neurofibromas and in those with large number of cutaneous (> 1,000) or subcutaneous (> 100) neurofibromas than in patients without such tumours. Most notably, MIA serum levels correlated significantly with internal tumour burden.ConclusionsMIA is a potential serum biomarker of tumour load in NF1 patients which could be useful in following the disease course and monitoring the efficacy of therapies.