FGFR1 mutations cause Hartsfield syndrome, the unique association of holoprosencephaly and ectrodactyly

Abstract

Background: Harstfield syndrome is the rare and unique association of holoprosencephaly (HPE) and ectrodactyly, with or without cleft lip and palate, and variable additional features. All the reported cases occurred sporadically. Although several causal genes of HPE and ectrodactyly have been identified, the genetic cause of Hartsfield syndrome remains unknown. We hypothesised that a single key developmental gene may underlie the co-occurrence of HPE and ectrodactyly.

Methods: We used whole exome sequencing in four isolated cases including one case-parents trio, and direct Sanger sequencing of three additional cases, to investigate the causative variants in Hartsfield syndrome.

Results: We identified a novel FGFR1 mutation in six out of seven patients. Affected residues are highly conserved and are located in the extracellular binding domain of the receptor (two homozygous mutations) or the intracellular tyrosine kinase domain (four heterozygous de novo variants). Strikingly, among the six novel mutations, three are located in close proximity to the ATP's phosphates or the coordinating magnesium, with one position required for kinase activity, and three are adjacent to known mutations involved in Kallmann syndrome plus other developmental anomalies.

Conclusions: Dominant or recessive FGFR1 mutations are responsible for Hartsfield syndrome, consistent with the known roles of FGFR1 in vertebrate ontogeny and conditional Fgfr1-deficient mice. Our study shows that, in humans, lack of accurate FGFR1 activation can disrupt both brain and hand/foot midline development, and that FGFR1 loss-of-function mutations are responsible for a wider spectrum of clinical anomalies than previously thought, ranging in severity from seemingly isolated hypogonadotropic hypogonadism, through Kallmann syndrome with or without additional features, to Hartsfield syndrome at its most severe end.

Keywords: Clinical genetics; Developmental; Genetics.

Figure 1

FGFR1 mutations are found in patients with Hartsfield syndrome. (A) Pictures of three patients diagnosed with Hartsfield syndrome, showing the wide range of disease severity. (B) Identification of the N628K mutation in patient 5. The upper part shows the exome sequencing reads (horizontal grey bars with mismatching bases highlighted) aligned to chromosome 8. Vertical bars above the reads represent the total number of reads covering a specific position (visualisation from Integrative Genomics Viewer37). The identified mutation is covered by 83 reads. The lower part shows the corresponding Sanger sequencing chromatogram. (C) Schematic representation of a FGFR1 dimer bound with FGF1 and the positions of Hartsfield syndrome mutations. Homozygous mutations are marked with an asterisk. (D) Sanger sequencing of patients 1–6. Chromatograms show FGFR1 mutations in patients 1–6 with Hartsfield syndrome, along with their parents. Parents of patient 2 were unavailable. For each patient, the reference sequence from human genome GRCh37 surrounding the mutated position is shown on top, and the sequence from Sanger sequencing is shown below.

Figure 2

Mapping of mutations L165S and L191S on crystal structure. Protein Data Bank structure 3OJV showing the extracellular Ig-like domains 2 and 3 of FGFR1 (amino acids 147–359) bound to FGF1 in surface representation, and detail around leucine 165 in ribbon representation. FGFR1 is shown in grey and FGF1 in blue. Leucines 165 and 191 are coloured in orange red. The detailed view is highlighting the interface between FGFR1 and FGF1 around leucine 165. Tyrosine 30 on FGF1 forms hydrogen bonds with leucine 165 and alanine 167. Substitution of the leucine 165 by a serine should affect FGF binding. These pictures were made using  UCSF Chimera.

Figure 3

Mapping of mutations G490R, D623Y, N628K, and C725Y on FGFR1 tyrosine kinase domain crystal structure. Protein Data Bank structure 3GQI showing the intracellular kinase domain of FGFR1 (residues 464–770) in ribbon representation. The lower left part shows the details of the crystal structure surrounding the ATP binding pocket in the intracellular kinase domain of FGFR1. G490, D623, and N628 are in close proximity to the ATP's phosphates or coordinating magnesium. The lower right part shows the involvement of cysteine 725 in the positioning of the αG-containing segment, along with T726, P722, and K721. Substitution of the cysteine 725 by a tyrosine will likely affect the conformation of this region. The ATP analogue (AMPPCP) and wild-type residues of positions 490, 623, 628 and 721, 722, 725 and 726 are pictured in stick representation. Nitrogen, oxygen, phosphorus, and magnesium atoms are coloured blue, red, orange, and green, respectively. These pictures were made using UCSF Chimera.

Figure 4

Mapping of FGFR1 mutations on known crystallographic structures. Idiopathic hypogonadotropic hypogonadism and Kallmann syndrome (KS) variants are depicted in black, KS with orofacial features variants are depicted in dark blue, more syndromic KS variants are depicted in magenta, and Hartsfield syndrome variants are depicted in orange red with the wild-type side chain in stick representation. Variants from the phenotypic three most severe categories are labelled. The most severe phenotype was considered if the same mutation was identified in several patients. Asterisk indicates a homozygous mutation. (A) Protein Data Bank (PDB) structure 3OJV, showing extracellular immunoglobulin (Ig)-like domains 2 and 3, from amino acids 147–359. (B) PDB structure 3GQI, showing the intracellular kinase domain, from residue 464–770. These pictures were made using UCSF Chimera.