Hum Genet DOI 10.1007/s00439-017-1804-9 ORIGINAL INVESTIGATION A rare variant in the FHL1 gene associated with X‑linked recessive hypoparathyroidism Nir Pillar1 · Oren Pleniceanu1,2 · Mingyan Fang3 · Limor Ziv4 · Einat Lahav2 · Shay Botchan1 · Le Cheng3 · Benjamin Dekel1,2 · Noam Shomron1 Received: 20 December 2016 / Accepted: 17 April 2017 © The Author(s) 2017. This article is an open access publication Abstract Isolated familial hypoparathyroidism is an c.C283T, p.R95W). Since FHL1 has not been linked to cal- extremely rare disorder, which to date has been linked cium regulation before, we provide evidence for its func- to several loci including mutations in CASR, GCM2, and tional role in hypoparathyroidism by: (i) bioinformatics PTH, as well as a rare condition defned as X-linked reces- analysis coupling its action to known modulators of PTH sive hypoparathyroidism, previously associated with a function; (ii) observing strong expression of fhl1b in Cor- 1.5 Mb region on Xq26-q27. Here, we report a patient with puscles of Stannius, gland-like aggregates in zebrafsh that hypocalcemia-induced seizures leading to the diagnosis of function in calcium regulation similar to mammalian PTH; primary hypoparathyroidism. Mutations in CASR, GCM2, and (iii) implicating fhl1b and FHL1 as regulators of cal- and PTH were ruled out, while whole exome sequencing of cium homeostasis in zebrafsh and human cells, respec- the family suggested FHL1, located on chromosome Xq26, tively. Altogether, our data suggest that FHL1 is a novel as the most likely causative gene variant (FHL1, exon 4, regulator of calcium homeostasis and implicate it as the causative gene for X-linked recessive hypoparathyroidism. Nir Pillar, Oren Pleniceanu, and Mingyan Fang are equal frst authors. Introduction Le Cheng, Benjamin Dekel, and Noam Shomron are equal last authors. Deep Sequencing, or Next Generation Sequencing (NGS), is a powerful tool to identify causative variants in clinical Electronic supplementary material The online version of this article (doi:10.1007/s00439-017-1804-9) contains supplementary cases, where other methods have been exhausted. Here, we material, which is available to authorized users. describe a 4-year-old male, born at full term following an uneventful pregnancy and delivery to non-consanguineous * Le Cheng parents, and four siblings (Fig. 1) presented with gen- [email protected] eralized seizure at the age of 9 days. Upon ruling out all * Benjamin Dekel other major etiologies for seizures, initial evaluation iden- [email protected] tifed severe hypocalcemia of 5.7 mg/dl [9–11], hyper- * Noam Shomron phosphatemia of 11.5 mg/dl [5–9], and inappropriately [email protected] low PTH of 20.7, alongside normal albumin, magnesium, 1 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, potassium, chloride, blood gases, and vitamin D levels. Israel Urinary calcium levels were low at 0.18 mg/dl [5.2–35.7]. 2 Pediatric Stem Cell Research Institute & Division Physical examination revealed no dysmorphic features, of Pediatric Nephrology, Edmond & Lily Safra Children’s short stature, malformations, or developmental delay. Addi- Hospital, Sheba Medical Center, Tel Hashomer, Israel tional studies included chest X-ray, brain US, echocardio- 3 BGI-Yunnan, Kunming, China gram, renal ultrasound, electroencephalogram, bone age 4 Sheba Cancer Research Center, Sheba Medical Center, study, and audiogram were all normal. Following a clinical Tel Hashomer, Israel geneticist consultation, 22q11.2 deletion was ruled out via 1 3 Hum Genet Fig. 1 Family pedigree. Squares denote male family members, circles female members, and shaded symbols affected members; the arrow points to the proband Fluorescent In-Situ Hybridization (FISH). Sanger Sequenc- with low allele coverage (<5 ) was detected. When focus- × ing detected no genomic variants in PTH and GCM2 genes. ing on homozygous recessive/X-linked variants, four vari- Since no additional loci were related to the phenotype, ants remained. Prioritization of these variants by combining array-CGH was not performed. variant severity and gene information revealed an X-linked At the age of 4 years, the patient continued to exhibit nonsynonymous mutation in FHL1, ‘Four and a Half LIM persistent hypocalcemia alongside inappropriately low domains 1’ gene (FHL1, exon 4, c.C283T, p.R95W) to PTH levels. In addition, mild orolingual muscle weak- be the top candidate variant. The variant was predicted to ness was detected in the patient. Notably, a younger, sixth be deleterious by the highest number of prediction tools female sibling was also found to have mild hypoparathy- (8.5/10; see “Materials and methods”). It is a very rare vari- roidism albeit without any electrolyte abnormality, while ant with prevalence of 0.0005/45 in the Exome Aggrega- all other family members exhibit normal PTH and calcium tion Consortium, 0.0006/6 in the NHLBI exome sequenc- levels. The patient’s father, as well as all other male sib- ing project and is not present in the 1000 genomes project, lings, demonstrates slight hypotonicity, requiring occupa- or is it found in our personal database of over 900 Israeli tional therapy. exomes. Sanger sequencing confrmed the variant is present Taken together, these fndings indicate that the patient in the patient, heterozygous in his mother, and not detected suffers from isolated familial hypoparathyroidism of in other family members. unknown origin. Exome sequencing of the affected male, The FHL1 c.C283T variant was predicted by the major- his parents, and siblings was performed to detect a causal ity of our employed tools to be deleterious, it is found in a gene. conserved region of the gene, and alternate allele coverage was >30 . Manually reviewing the three other homozy- × gous recessive/X-linked variants (ZNF366 c.A58G p.K20E, Results CORO1B c.G1252A p.A418T, and ZNF208 c.T986C p.I329T), all three variants were not predicted to be delete- Genomic analysis uncovers FHL1 as the causative gene rious by any of the tools we employed. FHL1 (also known as SLIM, SLIM1, or SLIMMER) is Whole exome sequencing (WES) of the patient generated a member of the gene family encoding LIM domain con- 1,065,106 variants passing initial flters, 58,977 of which taining proteins. An LIM domain is mainly constituted of were rare variants (allele frequency <1% in all databases), two cysteine-rich zinc-fnger motifs, which coordinately and 1031 were found inside exons and resulted in an amino bind zinc atoms to mediate protein–protein interactions acid change. When searching for de novo mutations found (Kadrmas and Beckerle 2004). FHL1 expression is highly in the patient or in his parents/siblings, only one variant enriched in striated muscles (Fig. 2), and has, therefore, 1 3 Hum Genet Fig. 2 Expression of FHL1 protein in human tissues, estimated by immunohistochemistry (IHC) using FHL1 antibody. Height of the bars repre- sents IHC staining strength. Taken from the Human Protein Atlas (Uhlén et al. 2015) been suggested to play an important role in skeletal mus- four of the manually curated targets plus FHL1 (Supple- cle growth and remodeling (Cowling et al. 2008). FHL1 was mentary Table 1). Next, to test the interactions between demonstrated to have an important role in muscle devel- FHL1 and hypocalcemia-related proteins, we have manu- opment and disease. A number of genetic studies linked ally assembled a list of 11 genes (PTH, CALC1, PTHRP, FHL1 missense mutations to congenital myopathies previ- TBX1, GCM2, CASR, AIRE, GNA11, GATA3, GNAS, and ously recognized by particular structural features, including TRPM6), which code for proteins known to be involved in Reducing body myopathy (RBM) and Emery-Dreifuss Mus- calcium sensing/metabolism with direct connection to pri- cular Dystrophy (EDMD) (Cowling et al. 2011). Mutations mary hypoparathyroidism. in the FHL1 gene have also been associated with arrhyth- We have assembled an induced network module, con- mias (San Román et al. 2016), HCM (Xu et al. 2015), and sisting of the 11 proteins coded by hypocalcemia-related dilated cardiomyopathy (Christodoulou et al. 2014) in sev- genes and FHL1 (Fig. 3). The induced network demon- eral patients affected by skeletal muscle disorders as well. strated an indirect connection between FHL1 and GATA3 Although the patient exhibits minimal muscular symp- (via STAT4) and CASR (via AKAP12 and FLNA). toms (see above), the phenotype differs signifcantly than STAT4 is a transcription factor belonging to the STAT previously ascribed to FHL1 mutations. Nonetheless, due protein family that is expressed mostly in immune cells and to the clinical assessment that the patient’s severe pheno- binds to hundreds of sites in the genome. It was shown that type and early onset strongly suggest a genetic cause, we FHl1 promotes both the degradation and the dephosphoryl- have decided to further pursue the connection between ation of STAT4 (Tanaka et al. 2005), presumably affecting FHL1 and hypocalcemia. GATA3 expression via this mechanism. AKAP12 (A-kinase anchor proteins) is a member of FHL1 interacts with calcium‑regulatory proteins a structurally diverse protein group, which has the com- mon function of binding to the regulatory subunit of pro- Using gene ontology, we tested the biological pathways tein kinase A (PKA) and confning the holoenzyme to dis- that were enriched by some of the 11 core genes plus crete locations within the cell. A connection with FHL1 FHL1. Several pathways associated with molecules trans- was noted in direct protein interaction (Vinayagam et al. port, including ion transport, transmembrane transport, 2011) and FLNA (Malovannaya et al. 2011). FLNA, coded and regulation of transport, were enriched with at least for Filamin A protein, is known to interact with CASR 1 3 Hum Genet Fig. 3 Induced module of FHL1 and connected proteins of interest. Nodes with black labels are seed proteins, and nodes with gray labels are intermediate nodes. Taken from ConsensusPathDB (Kamburov et al.