Genomic analysis uncovers FHL1 as the causative gene
Whole exome sequencing (WES) of the patient generated 1,065,106 variants passing initial filters, 58,977 of which were rare variants (allele frequency <1% in all databases), and 1031 were found inside exons and resulted in an amino acid change. When searching for de novo mutations found in the patient or in his parents/siblings, only one variant with low allele coverage (<5×) was detected. When focusing on homozygous recessive/X-linked variants, four variants remained. Prioritization of these variants by combining variant severity and gene information revealed an X-linked nonsynonymous mutation in FHL1, ‘Four and a Half LIM domains 1’ gene (FHL1, exon 4, c.C283T, p.R95W) to be the top candidate variant. The variant was predicted to be deleterious by the highest number of prediction tools (8.5/10; see “Materials and methods”). It is a very rare variant with prevalence of 0.0005/45 in the Exome Aggregation Consortium, 0.0006/6 in the NHLBI exome sequencing project and is not present in the 1000 genomes project, or is it found in our personal database of over 900 Israeli exomes. Sanger sequencing confirmed the variant is present in the patient, heterozygous in his mother, and not detected in other family members.
The FHL1 c.C283T variant was predicted by the majority of our employed tools to be deleterious, it is found in a conserved region of the gene, and alternate allele coverage was >30×. Manually reviewing the three other homozygous recessive/X-linked variants (ZNF366 c.A58G p.K20E, CORO1B c.G1252A p.A418T, and ZNF208 c.T986C p.I329T), all three variants were not predicted to be deleterious by any of the tools we employed.
FHL1 (also known as SLIM, SLIM1, or SLIMMER) is a member of the gene family encoding LIM domain containing proteins. An LIM domain is mainly constituted of two cysteine-rich zinc-finger motifs, which coordinately bind zinc atoms to mediate protein–protein interactions (Kadrmas and Beckerle 2004). FHL1 expression is highly enriched in striated muscles (Fig. 2), and has, therefore, been suggested to play an important role in skeletal muscle growth and remodeling (Cowling et al. 2008). FHL1 was demonstrated to have an important role in muscle development and disease. A number of genetic studies linked FHL1 missense mutations to congenital myopathies previously recognized by particular structural features, including Reducing body myopathy (RBM) and Emery-Dreifuss Muscular Dystrophy (EDMD) (Cowling et al. 2011). Mutations in the FHL1 gene have also been associated with arrhythmias (San Román et al. 2016), HCM (Xu et al. 2015), and dilated cardiomyopathy (Christodoulou et al. 2014) in several patients affected by skeletal muscle disorders as well.
Although the patient exhibits minimal muscular symptoms (see above), the phenotype differs significantly than previously ascribed to FHL1 mutations. Nonetheless, due to the clinical assessment that the patient’s severe phenotype and early onset strongly suggest a genetic cause, we have decided to further pursue the connection between FHL1 and hypocalcemia.
FHL1 interacts with calcium-regulatory proteins
Using gene ontology, we tested the biological pathways that were enriched by some of the 11 core genes plus FHL1. Several pathways associated with molecules transport, including ion transport, transmembrane transport, and regulation of transport, were enriched with at least four of the manually curated targets plus FHL1 (Supplementary Table 1). Next, to test the interactions between FHL1 and hypocalcemia-related proteins, we have manually assembled a list of 11 genes (PTH, CALC1, PTHRP, TBX1, GCM2, CASR, AIRE, GNA11, GATA3, GNAS, and TRPM6), which code for proteins known to be involved in calcium sensing/metabolism with direct connection to primary hypoparathyroidism.
We have assembled an induced network module, consisting of the 11 proteins coded by hypocalcemia-related genes and FHL1 (Fig. 3). The induced network demonstrated an indirect connection between FHL1 and GATA3 (via STAT4) and CASR (via AKAP12 and FLNA).
STAT4 is a transcription factor belonging to the STAT protein family that is expressed mostly in immune cells and binds to hundreds of sites in the genome. It was shown that FHl1 promotes both the degradation and the dephosphorylation of STAT4 (Tanaka et al. 2005), presumably affecting GATA3 expression via this mechanism.
AKAP12 (A-kinase anchor proteins) is a member of a structurally diverse protein group, which has the common function of binding to the regulatory subunit of protein kinase A (PKA) and confining the holoenzyme to discrete locations within the cell. A connection with FHL1 was noted in direct protein interaction (Vinayagam et al. 2011) and FLNA (Malovannaya et al. 2011). FLNA, coded for Filamin A protein, is known to interact with CASR (calcium-sensing receptor) and likely act as part of a scaffold that binds signaling components and other key cellular elements (e.g., the cytoskeleton) to facilitate the interaction of the receptor with its signaling pathways (Ray 2015).
fhl1b regulates calcium levels in zebrafish
The zebrafish (Danio rerio) is a powerful model organism for studying vertebrate biology, being well suited to both developmental and genetic analysis (Dooley and Zon 2000). Regulation of Ca2+ levels in vertebrates requires the ability to sense extracellular Ca2+ concentrations, a process carried out by the transmembrane calcium-sensing receptor (CASR) (Loretz 2008). Low Ca2+ levels sensed by CASR in the parathyroid gland, the main organ responsible for calcium homeostasis, lead to expression and secretion of PTH, the key hypercalcemic hormone, which acts on the kidneys, bones, and intestine to increase calcium levels. In contrast, lower vertebrates, like fish rely mostly on gills (or skin, during embryonic stages) as the primary organs for Ca2+ uptake (Hwang et al. 2011; Liao et al. 2007). Nonetheless, although fish do not harbor parathyroid glands, similar calcium-regulating pathways have been uncovered, involving mainly the Corpuscles of Stannius (CS), gland-like aggregates adjacent to the fish kidneys. CS strongly express CASR, which mediates the expression of PTH1 and STC1 (Stanniocalcin), the main hypercalcemic, and hypocalcemic hormones in fish, respectively (Hwang and Chou 2013). Thus, zebrafish represent an important model for studying calcium homeostasis in both physiological and disease states.
Similar to many other genes, due to teleost genome duplication, FHL1 is represented in fish by two paralogs: fhl1a and fhl1b (Glasauer and Neuhauss 2014). In light of its human phenotype, most studies to date have focused on the role of fhl1a and fhl1b on muscular and cardiac function (Bührdel et al. 2015; Chauvigné et al. 2005; Li et al. 2013) and more recently (Xu et al. 2016) on its role in pancreas-liver fate decision during development. Accordingly, in-situ hybridization experiments have demonstrated (Thisse et al. 2015) expression of fhl1a and fhl1b in the zebrafish muscular system, heart and pancreas, among others. Interestingly, fhl1b has been shown to be expressed strongly and specifically in the CS. Hence, we hypothesized that fhl1b might be involved in calcium regulation in fish.
To test this hypothesis, we injected fish embryos with morpholinos to inhibit the translation of zebrafish fhl1b and compared free plasma calcium levels between morpholinos-injected (fhl1b ATG MO) and uninjected (UI) fish. fhl1b expression was decreased by 45% in ATG MO group (SEM—3.2%, P < 0.01). The analysis was carried out in fish grown in media containing both low and high physiologic (25 μg/ml) calcium levels, so as to evaluate the potential relevance of fhl1b to calcium homeostasis across a wide range of calcium levels.
We detected an increase in plasma calcium levels in fhl1b ATG MO fish compared UI fish in both low and high calcium media (P < 0.05 for fish grown in low calcium media and P = 0.12 for 25 μg/ml Calcium media) (Fig. 4). Taken together, these results indicate a role for fhl1b in regulating fish plasma calcium levels.
fhl1b expression affects other calcium-regulatory genes
Having demonstrated the significant effect of fhl1b down-regulation on zebrafish plasma calcium levels, we were next interested in determining the mediators of this effect. Towards this end, we compared the expression of key calcium-regulatory genes (parathyroid hormone paralog 1—PTH1, calcium-sensing receptor—CASR, Stanniocalcin—STC1, epithelial calcium channel—ECaC, and GATA Binding Protein 3-Gata3) in fhl1b ATG MO fish to UI fish via quantitative RT-PCR (qPCR) (Fig. 5). Consistent with the increased calcium levels observed in fhl1b ATG MO fish, we detected in the latter significant up-regulation of PTH1, the main hypercalcemic hormone in both fish and mammals, compared to UI fish. Interestingly, we also found induction of CASR transcript levels in fhl1b ATG MO fish. All other genes were not affected by fhl1b down-regulation (Fig. 5). Hence, increased calcium levels detected in fhl1b-deficient fish are associated with increased PTH1/CASR expression.
fhl1b is a positive regulator of PTH expression in human cells
To assess a potential direct effect of FHL1 on calcium regulation via PTH expression in human cells, we used a luciferase reporter assay, whereby human embryonic kidney (HEK293) cells were transfected with a construct containing the luciferase reporter under the control of the human PTH promoter. Activation of PTH locus was measured by bioluminescence in the presence of empty vector or FHL1, with and without CASR presence, under both normal (1.8 mM calcium) and high calcium conditions (3.6 mM calcium). We detected a strong and significant increase in activation of the PTH promoter when FHL1 was introduced into the cells, in both normal and high calcium conditions (Fig. 6). Next, activation of PTH locus was measured by bioluminescence in the presence of siRNA against FHL1 or control siRNA. A consistent decrease in PTH activation was noted in the FHL1 siRNA group which was more significant in the high calcium conditions (Fig. 7). Taken together, these results indicate that FHL1 might function as a positive regulator of PTH expression in human cells.