Magdalena Socha

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.

Three novel GJA1 missense substitutions resulting in oculo-dento-digital dysplasia (ODDD) — Further extension of the mutational spectrum

Oculodentodigital dysplasia (ODDD) is a clinically variable genetic disorder caused by mutations of the GJA1 gene, predominantly inherited in an autosomal dominant fashion. In rare cases ODDD can also exhibit autosomal recessive mode of inheritance. The phenotype of ODDD comprises craniofacial (short and narrow palpebral fissure, thin, narrow nose with hypoplastic alae nasi), dental (oligodontia, hypoplastic enamel), and digital abnormalities (syndactyly of finger 4/5, hypoplastic phalanges). Ocular manifestation is typical and involves microphthalmia, microcornea, glaucoma, congenital malformations of iris or vitreous, ectopic pupils or strabismus. To date, only 67 GJA1 mutations have been described to underlie ODDD and most of them (i.e. 97%) represent missense substitutions. In this report, we describe three (two familial and one sporadic) non-consanguineous cases presenting with ODDD features in whom we identified novel missense heterozygous mutations of the GJA1 gene: c.317T>G (p. L106R), c.G139C (p.D47H), and c.C257A (p.S86Y). The first two mutations were inherited from an affected parent, whereas the latter one occurred de novo. The mutations affect highly conserved amino acid residues located in the different portions of the GJA1 protein. Our report broadens the spectrum of probably pathogenic mutations associated with ODDD phenotype and demonstrates that the amino acid substitutions at highly conserved positions 47, 86, 106 may affect protein functioning and lead to the development of this syndrome. Together with molecular data, we provide a brief clinical description of the affected individuals.

Variable expressivity of the phenotype in two families with brachydactyly type E, craniofacial dysmorphism, short stature and delayed bone age caused by novel heterozygous mutations in the PTHLH gene

Brachydactyly refers to shortening of digits due to hypoplasia or aplasia of bones forming the hands and/or feet. Isolated brachydactyly type E (BDE), which is characterized by shortened metacarpals and/or metatarsals, results in a small proportion of patients from HOXD13 or PTHLH mutations, although in the majority of cases molecular lesion remains unknown. BDE, like other brachydactylies, shows clinical heterogeneity with highly variable intrafamilial and interindividual expressivity. In this study, we investigated two Polish cases (one familial and one sporadic) presenting with BDE and additional symptoms due to novel PTHLH mutations. Apart from BDE, the affected family showed short stature, mild craniofacial dysmorphism and delayed bone age. Sanger sequencing of PTHLH revealed a novel heterozygous frameshift mutation c.258delC(p.N87Tfs*18) in two affected individuals and one relative manifesting mild brachydactyly. The sporadic patient, in addition to BDE, presented with craniofacial dysmorphism, normal stature and bone age, and was demonstrated to carry a de novo heterozygous c.166C>T(p.R56*) mutation. Our paper reports on the two novel truncating PTHLH variants, resulting in variable combination of BDE and other symptoms. Data shown here expand the knowledge on the phenotypic presentation of PTHLH mutations, highlighting significant clinical variability and incomplete penetrance of the PTHLH-related symptoms.

Further evidence for FGF16 truncating mutations as the cause of X-linked recessive fusion of metacarpals 4 / 5

BACKGROUND Metacarpal 4-5 fusion (MF4; MIM#309630) is a rare congenital malformation of the hand characterized 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. Recently, we have identified FGF16 nonsense mutations as the underlying cause of isolated X-linked recessive MF4. METHODS In this report, we provide a detailed clinical description of a sporadic male patient showing MF4 in whom we performed Sanger sequencing of the entire coding sequence of FGF16. RESULTS In addition to MF4 symptoms, the patient presented with generalized joint laxity and hypermobility. FGF16 sequencing detected a novel truncating mutation (c.474_477del; p.E158DfsX25) in exon 3 of the gene. A heterozygous mutation was found in a clinically and radiologically unaffected mother of the proband. CONCLUSION Our finding confirms that truncating mutations of FGF16 are causative for X-linked recessive metacarpal 4-5 fusion. Importantly, the mutation detected in this study was located in last exon of the gene (exon 3), like the only two FGF16 disease-causing variants identified to date. Thus, all FGF16 mutations known to give rise to this rare skeletal hand malformation are C-terminal and most probably do not result in a nonsense mediated decay. Additionally, our proband showed mild symptoms of a connective tissue disorder, as some other patients previously reported to have X-linked MF4. Therefore, we suggest that impaired FGF16 function may also be responsible for connective tissue symptoms in MF4 patients.

Noncoding copy-number variations are associated with congenital limb malformation

PurposeCopy-number variants (CNVs) are generally interpreted by linking the effects of gene dosage with phenotypes. The clinical interpretation of noncoding CNVs remains challenging. We investigated the percentage of disease-associated CNVs in patients with congenital limb malformations that affect noncoding cis-regulatory sequences versus genes sensitive to gene dosage effects.MethodsWe applied high-resolution copy-number analysis to 340 unrelated individuals with isolated limb malformation. To investigate novel candidate CNVs, we re-engineered human CNVs in mice using clustered regularly interspaced short palindromic repeats (CRISPR)–based genome editing.ResultsOf the individuals studied, 10% harbored CNVs segregating with the phenotype in the affected families. We identified 31 CNVs previously associated with congenital limb malformations and four novel candidate CNVs. Most of the disease-associated CNVs (57%) affected the noncoding cis-regulatory genome, while only 43% included a known disease gene and were likely to result from gene dosage effects. In transgenic mice harboring four novel candidate CNVs, we observed altered gene expression in all cases, indicating that the CNVs had a regulatory effect either by changing the enhancer dosage or altering the topological associating domain architecture of the genome.ConclusionOur findings suggest that CNVs affecting noncoding regulatory elements are a major cause of congenital limb malformations.

Clinical and molecular genetic characterization of a male patient with Sensenbrenner syndrome (cranioectodermal dysplasia) and biallelic WDR35 mutations

Sensenbrenner syndrome (cranioectodermal dysplasia, CED) is a very rare autosomal recessive ciliopathy first described by Judith Sensenbrenner in 1975. CED is a complex disorder characterized by craniofacial, skeletal, and ectodermal abnormalities. The clinical symptoms are variable and the CED phenotype may present intrafamilial and interfamilial differences. Sensenbrenner syndrome belongs to a group of ciliary chondrodysplasias and is a genetically heterogeneous disease. Mutations in six genes: IFT122, WDR35, IFT43, WDR19, IFT52, and IFT140 have been associated with this disorder. All known CED genes encode proteins that are part of the intraflagellar transport complex, which plays an important role in the assembly and maintenance of cilia.

Prenatal diagnosis of Fraser syndrome using routine ultrasound examination, confirmed by exome sequencing – report of a novel homozygous missense FRAS1 mutation

Current clinical diagnostic criteria of FS proposed by Van Haelst et al. (2007) include six major symptoms (cryptophthalmos spectrum, syndactyly, ambiguous genitalia, urinary tract abnormalities, laryngeal and tracheal anomalies, and positive family history) and five minor symptoms (anorectal malformation, skull ossification defects, dysplastic ears, umbilical defects, and nasal anomalies). FS can be clinically recognized if either three major criteria, or two major and two minor criteria, or one major and three minor symptoms are present in a patient (van Haelst et al. 2007). About half of the cases presenting with classical phenotype of FS are caused by FRAS1 mutations (OMIM 607830), while some other result from the FREM2 (OMIM 608945) or GRIP1 (OMIM 604597) gene lesions (McGregor et al. 2003; Jadeja et al. 2005; Shafeghatti et al. 2008; Vogel et al. 2012; Hoefele et al. 2013). In this paper, we report on prenatal presentation of the Fraser syndrome in a patient with multiple congenital anomalies. Wholeexome sequencing (WES) confirmed the diagnosis and demonstrated a novel homozygous causative missense mutation in FRAS1. We investigated a Polish family, in which three out of five children showed clinical symptoms of FS (for pedigree, see Fig. 1A). Molecular analysis was initiated in one of the affected offspring (index case II-5 born from G5), prenatally diagnosed with multiple congenital anomalies. In addition, we studied two unaffected brothers and both healthy parents of the proband. The proband was conceived by a healthy consanguineous couple (second cousins once removed), after four previous pregnancies. Their first and second pregnancy (II-1 and II-2) resulted in live birth of a male and a female newborn, respectively, who died a few hours after birth due to multiple congenital anomalies. The karyotype of the second child tested on the basis of fetal amniocytes was normal. Since DNA of both affected children was unavailable, genetic testing was not performed. The third and fourth pregnancies were uneventful and resulted in delivery of two healthy males (II-3 and II-4). In the fifth pregnancy, the 20-weeks ultrasound showed large, hyperechoic lungs of the fetus (Fig. 1B), pathognomonic for CHAOS (Congenital High Airway Obstruction Syndrome). The next fetal scan revealed additional anomalies including asymmetry of orbits, facial dysmorphism, including broad nasal bridge, and a suspicion of syndactyly. The parents decided to continue the pregnancy. The proband was born at 38weeks of gestation with body weight of 3590 g and died 1 h after birth due to multiple congenital anomalies, including laryngeal atresia. All other prenatally diagnosed or suspected abnormalities were confirmed after birth, including the complete syndactyly of both hands and feet and left cryptophthalmos. Due to ambiguous genitalia, phenotypic sex of the child was difficult to recognize. In order to confirm the clinical diagnosis of FS in our proband, we performedWES on a single DNA sample of the index patient (II-5). A novel homozygous missense FRAS1 variant c.10311C>G(p. Cys3437Trp) in exon 69 was identified (Fig. 1C). Sanger sequencing confirmed WES findings in the proband and demonstrated that both parents as well as their two healthy sons were the carriers of the heterozygous c.10311C>G(p.Cys3437Trp) alteration (Fig. 1D). The variant was predicted to be pathogenic by PolyPhen2 (1.0 score), Mutation Taster2 (1.0), and SIFT (0.0 score).

Clinical expression of Holt-Oram syndrome on the basis of own clinical experience considering prenatal diagnosis

OBJECTIVES Holt-Oram syndrome manifests with defects of upper limbs, pectoral girdle and cardiovascular system. The aim of this paper was to present complex clinical picture of the syndrome and its variable expression on the example of the family diagnosed genetically on the neonatal ward, after probands prenatal examination. MARETIAL AND METHODS Nine family members were tested for TBX5 gene mutation. RESULTS Four of family members were diagnosed with Holt-Oram syndrome and five had correct genetic test results. The diagnosis allowed to identify a genetic risk family and enabled to provide them with genetic counselling. CONCLUSIONS Diagnosis of Holt-Oram syndrome is possible as early as in prenatal period and it can be verified by genetic tests.