FLD and FED are rare LCAT deficiency syndromes with differing clinical manifestations. In this article, we discuss the results of the first systematic analysis evaluating the ethnic distribution of LCAT deficiency, with particular emphasis on Latin America, and we present the case histories of three Mexican-Mestizo probands.
The systematic review retrieved 215 published cases of which 71.6% were reported as FLD, 19% as FED and 9.3% were unclassified. This number is significantly greater than that reported in the current literature (101). The majority of probands have been published in case reports, often with incomplete clinical or genetic information. It is evident that this disease continues to be encountered at a late age, with corneal opacifications being the principal reason for consultation. Furthermore, FED is diagnosed significantly later than FLD, probably due to the more severe clinical phenotype of the later warranting earlier medical attention. Timely diagnosis of this disease is needed for the application of preventive strategies and the use of newer therapies.
The biochemical features of the cases showed that LCAT enzyme activity was significantly lower in FLD compared to FED. Low HDL-C levels are a characteristic feature of this disease, however, there was no difference in concentrations between the phenotypes. Indeed, FLD and FED can have similar lipid profiles, suggesting any variability in parameters is unrelated to LCAT function. Pavanello et al. have commented that the severity of the hypoalphalipoproteinemia varies widely among carriers of different LCAT genotypes (101). Furthermore, carriers of one mutant LCAT allele show an intermediate biochemical phenotype between homozygous carriers and controls, suggesting that the disease, which is reported as recessive, is indeed co-dominant for the biochemical phenotype. This was also confirmed in the relatives of the Mexican-Mestizo probands.
The clinical features of the cases showed a clear difference between FED and FLD, with renal disease and anemia prevalent in the later. However, not all the FLD cases showed significant proteinuria or a reduction in eGFR; this suggests that the rate of progression of renal failure may well be variable. Lamiquiz-Moneo et al. state that this clinical variability is likely to be related to the biochemical phenotype rather than to the inherited mutation (80). In addition, 9.3% of cases had an unclassified clinical phenotype; the authors could not confirm either familial LCAT deficiency (FLD) or fish eye disease (FED). Some authors have commented that the clinical manifestations of patients with LCAT gene mutations may vary even among members of the same family carrying identical mutations (41). Therefore, it is evident that LCAT deficiency syndromes show both biochemical and clinical heterogeneity.
An important finding of this systematic review was the significantly greater prevalence of premature CHD in FED patients compared to FLD patients. The cardiovascular risk associated with LCAT deficiency syndromes has been a matter of debate for a number of years. A severe deficiency of HDL-C in LCAT deficient carriers would be expected to increase their risk of developing coronary heart disease (83). Oldoni et al., have compared carotid intima media thickness between 33 heterozygous FLD subjects and 41 heterozygous FED subjects (102). Carriers of FLD mutations exhibited less carotid atherosclerosis, whereas those with FED mutations presented with more subclinical atherosclerosis. The authors proposed that this discrepancy was related to the capacity of LCAT to esterify cholesterol on apolipoprotein B–containing lipoproteins- this capacity is lost in FLD, but is unaffected in FED. In a study of Italian FLD families, the inheritance of a mutated LCAT genotype had a remarkable gene-dose dependent effect in reducing carotid IMT, whereas a subgroup of these carriers also showed normal flow-mediated dilation (65, 83, 103). A Mendelian randomization study in 54,500 subjects concluded that common genetic variation in LCAT resulting in decreased HDL-C levels, did not associate with an increased risk of ischemic cardiovascular disease (104). Low HDL-C levels robustly associated with increased risk of myocardial infarction (MI), but genetically decreased HDL cholesterol did not. This may suggest that isolated low HDL cholesterol levels do not cause MI; the inverse relation between HDL-C and CHD observed in epidemiological studies may not be causal.
The molecular defects associated with LCAT deficiency syndromes show heterogeneity. In total, 138 LCAT mutations were encountered with no particular exon dominating in a particular ethnicity. Again this number is greater than that reported in the literature (101). There was no association between clinical phenotype and genetic alteration, this may be due to the low number of cases worldwide. Exon 6 was the predominant site for both FLD and FED; however, after adjusting for exon size, exon 1 and 4 showed the greatest concentration of mutations. At present, it is impossible to predict the phenotype (FLD or FED) associated with the LCAT mutations (101).
With regards to ethnicity, at least 33 different groups are represented, of which Caucasians are most common. The predominance of Caucasian and Asian cases may reflect better health awareness and access to health care compared with developing regions. Remarkably, only one case has been found in the African sub-continent. Cases are more likely to be found in affluent countries, but also in countries hosting research groups with interest and resources to investigate this disease. In Latin American few cases have been published (82–88). At present, only six countries have reported probands with LCAT deficiency, and genetic evaluation has only been carried out in three countries. Of these, Brazil reports three mutations causing FLD each associated with a distinct geographical region. In Mexico, the first FLD proband, came from an isolated village in the south of Mexico, there is little genetic admixture in this region. The indigenous heritage of this patient may have been responsible for disease susceptibility. The mutations reported in Brazil and Chile are on exon 5 and 6. In Mexico, exon 1 mutations predominate (3 probands), with only 1 case from the north of the country showing a homozygous exon 4 alteration.
The first Mexican- Mestizo proband (FLD phenotype) showed an alteration located in the leader sequence (a stop codon at position 8 in exon 1 of the LCAT gene), thus normal protein synthesis is abolished. In accordance with the genetic results, the laboratory results confirmed low HDL-C concentrations and very low specific LCAT activity. Both parents and 2 siblings were heterozygotes and presented with intermediate low HDL-C levels (< 40 mg/dl) and low specific LCAT activity (22–36%). To our knowledge, this particular mutation is novel, only one other mutation in this region of the gene has been reported: Calabresi et al. mention a subject with a Thr-13Met mutation and an FLD phenotype (64).
On interesting finding was that the proband continued to show paroxonase- 1 activity. This was essentially on HDL, even though the number of particles was extremely low and despite a clear lack of LCAT activity. The proband had 27% activity, while her affected siblings had approximately 50% activity. The heterozygote family members had essentially normal paroxonase-1 (PON-1) activity. This enzyme prevents the conversion of LDL cholesterol into a more atherogenic particle (105). Preserved PON-1 activity has been reported in other HDL deficiency states, and in vitro experiments with LCAT deficient plasma suggest an apparent maintenance of cholesterol efflux (94, 106, 107). Although HDL cholesterol levels were reduced by 93%, there was only a 50% reduction in reverse cholesterol transport (RCT). This suggests that RCT is conserved even in the presence of complete LCAT deficiency, supporting the differential cardiovascular risk between phenotypes.
The second Mexican- Mestizo proband (FED) had two distinct LCAT mutations, one on each allele (compound heterozygote). One of the alterations was a frameshift mutation (c.101dupC) on exon 1; the other mutation was a missense mutation (c.110C > T) on the same exon. Both mutations have previously been reported in the literature (25, 26, 27).
Argyropoulos et al. have reported an FLD Caucasian proband who was compound heterozygote with a missense mutation identical to that in our proband (c.110C > T on exon 1) (44). Posadas-Sanchez et al. have reported the presence of the same missense mutation (c.110C > T) on both alleles (homozygote) in an unrelated Mexican subject with an unclassified LCAT deficiency syndrome (probable FED) (88). The 34- year old Mexican man had type 2 diabetes, premature coronary artery disease, corneal opacities, normal renal function and extremely low levels of HDL cholesterol (2 mg/dl). The investigators reported an increase in the number of small HDL particles in the proband. In addition, the HDL-C particles had a reduced ability to promote cholesterol efflux (PON-1 activity was low). Finally, Bujo et al. have published the presence of the homozygous c.101dupC mutation on exon 1 in a Japanese subject. This resulted in a truncated 16 amino-acid non -functional LCAT protein and an FLD phenotype (20).
Predicting the effect of the co- existence of these mutations (one on each allele) on LCAT function and structure is not straightforward. The majority of mutations are not located in sites involved in the catalytic function of the enzyme; the affected sites are probably involved in maintaining protein stability and structure. The mature LCAT protein contains 416 amino acids and a leader sequence (67 kDa) (108, 109). The enzyme is thought to undergo post- translational glycosylation which appears to be essential for the conformational stability of the protein (110). In addition, LCAT has two disulfide bridges between Cyst50-Cys74 and Cys313-Cys356; the first bridge partially covers the active site of LCAT, forms part of the lid region and is thought to enable the enzyme to bind to lipid surfaces. Hence, in this patient, the genetic alterations may interfere with the nearby lid structure or produce a conformational change when the mature protein is folded, resulting in enzyme- substrate interference. The frameshift mutation is a more detrimental alteration; however, clinical expression of which would only be apparent in homozygotes. Hence, the predominant phenotype in our subject is FED.
The third Mexican-Mestizo proband (FLD), had a point mutation on exon 4 of the LCAT gene, i.e., a G to A substitution on codon 140 converting Arginine to Histidine. This mutation has been reported previously in an Austrian kindred who were also homozygous for this modification (37). It appears that this domain (where Arg140 resides) is crucial for an enzymatically active LCAT protein, mutations in this region possibly affect tertiary structure.
In Latin America, persons with LCAT deficiency syndromes face unique challenges. The medical community is unaware of this condition; our third proband had attended consultations with several specialists, had undergone three kidney biopsies and one bone marrow aspiration; despite this, she had not been diagnosed. Additionally, many centers do not have the infrastructure to carry out the biochemical or genetic studies necessary to confirm this condition. Current management of FLD is preventative and involves lipid lowering therapy, ACE inhibitors, diuretics and steroids, in order to delay progression to end-stage renal disease: for many in Latin America, these medications will be an out of pocket expense. Furthermore, in this region, access to further treatment with peritoneal dialysis or hemodialysis is variable. This is related to fragmented health care coverage and socioeconomic inequality. Although, provision of renal replacement therapy (RRT) has increased in all Latin American countries over the past 20 years, universal access is available in only a few countries (Argentina, Brazil, Chile, Cuba, Uruguay, Venezuela, and Colombia) (111). Kidney transplantation may offer a temporary cure, but reoccurrence of nephropathy is inevitable and occurs within a few years (112). Currently, trials are underway with human recombinant LCAT enzyme and there is the possibility of gene therapy in the future (113). However, such products maybe subsequently unavailable and/or unaffordable (cost is much greater than average monthly income) to most of the Latin American population (114).
Earlier identification and adequate follow up of patients is urgently needed; the implementation of models of care and national disease registries can aid in this process. Recently, the Norwegian National Advisory Unit on Rare Disorders has been asked to establish a worldwide contact registry on FLD; this will allow the integration of efforts throughout the world to tackle the health burden and improve care for this condition (83).
We must acknowledge the strengths and limitations of this work. This is the first systematic review of LCAT deficiency syndromes; this work highlights the major knowledge gaps in this disease. The overall number of cases and mutations is far greater than currently thought. In Latin America, the limited number of cases may have influenced our findings and further work is necessary in order to confirm whether isolated geographical regions may have ethnicity specific mutations. Measurements of free cholesterol and cholesteryl ester, as well as cholesterol esterification rate to complete the biochemical characterization of the Mexican probands and their families would have been desirable, this was limited by the death of two of the probands, and the geographical location of the last proband.
In conclusion, the systematic review shows that LCAT deficiency syndromes are diagnosed late; with FLD cases identified significantly earlier than FED. Furthermore, we confirm that this condition is clinically and genetically heterogeneous. We were unable to confirm any association between ethnicity and LCAT mutation. However, we were able to show a significantly greater risk of premature coronary artery disease in FED compared to FLD. This finding is important, it suggests that management should be tailored according to the LCAT deficiency profile. In FLD patients, the priority is to mitigate progression to end stage kidney disease; in contrast, in FED patients, management of cardiovascular risk may well be paramount. Finally, the LCAT mutations discussed in this article are the only ones reported in the Mexican- Amerindian population. We report a novel mutation associated with FLD, in a Mexico-Mestizo woman, suggesting the influence of Amerindian ancestry.