GGCX mutations in a patient with PXE/CL-like overlapping phenotype

Summary

Pseudoxanthoma elasticum (PXE) is a multisystem disorder characterized by ectopic mineralization of connective tissues with primary manifestations in the skin, eyes, and cardiovascular system. The classic forms of PXE are caused by mutations in the ABCC6 gene encoding the ABCC6 protein expressed primarily in the liver. Cutis laxa (CL) manifests with loose and sagging skin with loss of recoil. In 2009 we investigated a 19-year-old patient with overlapping cutaneous features of PXE and CL, together with alpha thalassemia. Genetic analysis failed to identify pathogenic mutations in ABCC6. More recently we developed a gene-targeted panel of next-generation sequencing technology. This panel has 29 genes, 22 of them, including ABCC6 and GGCX, being associated with ectopic mineralization phenotypes. Mutation analysis identified two heterozygous GGCX mutations, c.200_201delTT in exon 2 and c.763G>A, p.V255M in exon 7. The GGCX gene encodes a γ-glutamyl carboxylase necessary for activation of blood coagulation factors in the liver. The p.V255M mutation was previously reported to result in reduced γ-glutamyl carboxylase activity in vitro while the c.200_201delTT mutation is novel. Previous studies reported that mutations in GGCX cause PXE/CL skin phenotypes in association with or without multiple vitamin K-dependent coagulation factor deficiency. Our patient had loose redundant skin, moderate to severe angioid streaks, characteristic calcification of elastic structures in mid-dermis, consistent with PXE/CL overlap, but no coagulation abnormalities. Our studies expand the GGCX mutation landscape in patients with PXE-like phenotypes.

Introduction

Pseudoxanthoma elasticum (PXE; OMIM #264800) is an autosomal recessive multisystem disorder with primary clinical manifestations in the skin, the eyes, and the arterial blood vessels.^1,2 The histopathological hallmark of PXE is ectopic calcium hydroxyapatite deposition in soft connective tissues, particularly the elastic structures. In the skin, the primary lesions are small, yellowish papules with predilection for flexural areas, and these lesions progressively coalesce into larger plaques of inelastic, leathery, and loose skin with yellowish hue. The skin histopathology reveals accumulation of pleiomorphic elastotic material in the upper and mid-dermis, which becomes progressively mineralized. The characteristic eye manifestations consist of angioid streaks. Mineralization of the elastin-containing retinal layer, the Bruch’s membrane, can result in fractures, neovascularization, and retinal bleeding causing progressive loss of visual acuity and central vision loss. Cardiovascular complications arise from the progressive mineralization of the elastin-rich arterial blood vessels, and clinical sequelae include intermittent claudication, renal hypertension, and, rarely, gastrointestinal bleeding, early myocardial infarction and stroke. The phenotypic spectrum of PXE is highly variable with both inter- and intrafamilial heterogeneity.

Classic PXE is caused by mutations in ABCC6, which encodes an efflux transporter protein ABCC6 primary expressed in the liver.² Hepatic ABCC6 with loss-of-function mutations results in reduced circulating concentrations of inorganic pyrophosphate, a potent anti-mineralization factor.² In addition to classic PXE, a number of genetically distinct clinical conditions display PXE-like clinical features with aberrant mineralization of elastic structures in the skin.² One of them involves mutations in GGCX encoding a γ-glutamyl carboxylase which catalyzes γ-carboxylation of glutamyl residues in several proteins including blood coagulation factors and matrix Gla protein (MGP), the latter serves as an anti-mineralization factor.³ Patients with GGCX mutations manifest with overlapping features of PXE and cutis laxa (CL) with or without vitamin K-dependent multiple coagulation factor deficiency.^4–7 These patients demonstrate cutaneous lesions that are similar to those in classic PXE, i.e., small yellowish papules, which tend to coalesce and which by histopathological and ultrastructural examination show profound mineralization. In addition, most patients with GGCX mutations demonstrate loose and redundant skin with loss of recoil, characteristic of CL.⁸

Case report

The patient was a 19-year-old Laotian man with a background of thalassemia, presented with a 4-year history of progressive asymptomatic skin laxity.⁹ The skin laxity was predominantly found in the flexural regions (including neck, axilla, inguinal regions) and abdomen with yellowish hue but lacking ‘plucked chicken-skin’ appearance (Fig. 1a). We therefore termed this condition “Pseudoxanthoma elasticum sine pseudoxanthoma”. There was no family history of PXE or PXE-like syndrome. Histopathological examination from skin biopsies highlighted using von Kossa staining showed mineral deposition in the mid dermis (Fig. 1b). Transmission electron microscopy revealed findings highly characteristic for PXE, including abnormal elastic fibers in the deep dermis and foci of ectopic mineralization (Fig. 1c).

Figure 1. Clinical features and mutation analysis of the patient.

(a) Yellowish papules, plaques, and loose and sagging skin on the back and in the axillary area. (b) von Kossa stain of the lesional skin revealed calcification of elastotic structures in the reticular dermis (arrows).⁹ (c) The lesional skin was processed into epoxy resin for transmission electron microscopy. The findings of abnormal elastic fibers in the mid dermis (asterisks) with foci of mineralization (arrow) are characteristic of PXE. (d) Ophthalmoscopic image of the retina revealed moderate to severe angioid streaks (arrows) in the right eye emanating around the optic nerve. (e,f) GGCX mutation analysis by Sanger sequencing. The patient was compound heterozygous for c.200_201delTT and c.763G>A mutations in the GGCX gene.

Echocardiogram was normal when he was 19, but at 21 years of age it showed an upper normal left ventricular end diastolic and end systolic dimension with a fractional shortening of 29% which is at the lower limit of normal. Chest x-ray and CT angiogram of the thoracic and abdominal vasculature and carotid and iliofemoral ultrasound studies were normal when he was 19 years old. The patient did not show the vascular signs or complications associated with PXE. However, given the diagnosis of a PXE-like syndrome an aggressive protection of his arteries from arteriosclerosis was suggested at age 19: a statin (atorvastatin) and an ACE inhibitor (perindopril arginine). An ophthalmologic examination revealed normal visual acuity but there was evidence of moderate to severe angioid streaks in the right eye (Fig. 1d) and very mild changes in the left eye. No evidence of retinal haemorrhages or alteration of retinal pigmentation on fundoscopy examination was noted.

The blood workup demonstrated largely normal mineral homeostasis, lipid profile, iron levels, rheumatoid factor latex, C reactive protein, thyroid stimulating hormone, and platelet studies (Table 1). The patient is asymptomatic for blood and metabolic disease and no evidence of clinically increased bleeding tendency was noted. Haemoglobin A2 was normal (2.1%, normal range 1.5%−3.1%). The low levels of hemoglobin (120 g/L, normal range 130–170 g/L), mean corpuscular volume (MCV) (54.8 fL, normal range 80–100 fL), and mean corpuscular hemoglobin (MCH) (17.1 pg, normal range 27–32 pg) as well as high red cell distribution width (RDW) (19.6%, normal range 11.6%−14%), suggested moderate to severe thalassemia (Table 1). Staining of blood cells revealed red cell inclusion bodies. In addition, gel electrophoresis in the alkaline pH showed two fast moving bands being the hemoglobin H and hemoglobin Bart’s. The blood cell count and hemoglobin workup (Table 1) suggested the diagnosis of hemoglobin H disease, a form of alpha thalassemia.

Table 1.

Blood chemistry of the patient

Tests	Analyte	Level	Units	Normal range
Mineral homeostasis	Calcium (Ca)	2.16	mmol/L	2.10–2.60
	Phosphate (Pi)	1.6	mmol/L	0.8–1.5
	Alkaline Phosphatase (ALP)	81	U/L	30–115
Lipid profile	Cholesterol	3.9	mmol/L	3.9–5.5
	Triglycerides	0.7	mmol/L	0.5–1.7
Iron studies	Ferritin	203	μg/L	20–400
	Iron	13	μmol/L	10–30
	Transferrin	2.0	g/L	1.8–3.2
	Transferrin Saturation	26	%	15–50
Blood cell count	White Blood Cell Count (WCC)	13.7	×109/L	4.0–10.5
	Red Blood Cell Count (RCC)	7.02	×1012/L	4.50–6.50
	Hemoglobin (Hb)	120	g/L	130–170
	Mean Corpuscular Volume (MCV)	54.8	fL	80.0–100.0
	Mean Corpuscular Hb (MCH)	17.1	pg	27.0–32.0
	Mean Corpuscular Hb Concentration (MCHC)	312	g/L	315–355
	Red Cell Distribution Width (RDW)	19.6	%	11.6–14.0
	Erythrocyte Sedimentation Rate (ESR)	5	mm/h	1–12
	Platelet (PLT)	266	×109/L	150–400
	Neutrophils	6.8	×109/L	2.0–7.0
	Lymphocytes	5.6	×109/L	1.0–3.0
	Monocytes	0.6	×109/L	0.2–1.0
	Eosinophils	0.7	×109/L	0.0–0.5
	Basophils	0.0	×109/L	0.0–0.1
Rheumatology studies	Rheumatoid Factor (RF) Latex	<7	IU/mL	0–14
Inflammation studies	C Reactive Protein (CRP)	<1	mg/L	0–10
Thyroid function studies	Thyroid Stimulating Hormone (TSH)	2.2	mU/L	0.3–5.0
Autoantibodies	Anti-Nuclear Abs (ANA)	Negative		Negative
	Anti-Extractable Nuclear Antigen Antibodies (ENA)	Negative		Negative
Hemoglobin (Hb) pattern analysis	Hemoglobin A2	2.10	%	1.5–3.1
	Hemoglobin F	2.7	%	0.3–4.4
	Hb H Bodies	Positive		Negative
	Variant Hb AlkEPG	Detected Hb H and Hb Bart’s		Negative

The results suggesting the diagnosis of alpha thalassemia are highlighted in bold. EPG, electrophoresis.

Mutation analysis by targeted exonic sequencing approach in 2009 failed to identify pathogenic mutations in ABCC6, the candidate gene for classic PXE. More recently, we developed a gene-targeted panel of next-generation sequencing platform targeting ectopic mineralization disorders. This panel has 29 genes, 22 of them, including ABCC6 and GGCX, are associated with ectopic mineralization phenotypes in human and/or mouse¹⁰. This sequencing panel also failed to detect mutations in ABCC6. Instead, mutation analysis in the patient identified two heterozygous GGCX variants, c.200_201delTT, p.F67Sfs*27 in exon 2 and c.763G>A, p.V255M in exon 7. Both variants were confirmed by Sanger sequencing (Fig. 1e,f). Bioinformatics analysis suggested their pathogenicity. Specifically, three commonly used, yet independent programs, MutationTaster, Polyphen-2, and SIFT, predicted both variants as pathogenic mutations. Next, we determined the Combined Annotation Depletion score (CADD) for each of the variants. CADD is a recently developed method for objective annotation of DNA sequence variants by combining more than 60 diverse prediction program with its own metrics into a single measure (CADD score).¹¹ The CADD scores for both variants were over 20, indicating the variants are among the 1% of most deleterious mutations to the human genome. In addition, the c.200_201delTT variant was not found in over 130,000 unrelated healthy individuals in the Genome Aggregation Database (gnomAD), and the c.763G>A variant was exceedingly rare in gnomAD database and no homozygous individuals were identified, suggesting that they are not frequent polymorphisms present in the general population. The p.V255M variant was previously reported to result in reduced γ-glutamyl carboxylase activity in vitro, supporting its pathogenecity⁶. The c.200_201delTT variant is predicted to cause out-of-frame translation and a premature stop codon, thus leading to synthesis of a shortened, nonfunctional GGCX protein (p.F67Sfs*27). The c.200_201delTT variant is a novel mutation, expanding the mutational landscape in GGCX.

PXE-like syndromes occurs in association with hemolytic anemias, in particular the hemoglobinopathies.¹² Patients with beta thalassemia often display PXE-like skin changes,¹³ yet without ABCC6 mutations.¹⁴ It was recently reported that PXE and beta thalassemia traits can co-occur on a genetic basis of variants in ABCC6 and other ectopic mineralization genes.¹⁵ The patient in this study had co-existent PXE/CL-like skin features and hemoglobin H disease, a form of alpha thalassemia, although ectopic mineralization and alpha thalassemia may be coincidental findings.

Bulleted points:

What’s already known about this topic?

• PXE is caused in most cases by mutations in the ABCC6 gene.

• Combined PXE and cutis laxa-like clinical findings have been encountered with vitamin K-dependent coagulation factor deficiency frequently caused by GGCX mutations.

• GGCX encodes a γ-glutamyl carboxylase necessary for activation of vitamin K-dependent coagulation factors in the liver.

What does this study add?

• This study reports a unique case with overlapping PXE/CL-like clinical phenotype with no evidence of a coagulation disorder.

• The patient carries compound heterozygous mutations in GGCX, adding to the mutation spectrum of GGCX-associated phenotypes.

Abbreviations:

CL

Cutis laxa

PXE

pseudoxanthoma elasticum