Hypobetalipoproteinemia
To view Lipoprotein Disorders Main Page Click here
To view Hypolipoproteinemia Main Page Click here
Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1] Associate Editor(s)-in-Chief: Aravind Kuchkuntla, M.B.B.S[2]
Synonyms and keywords: Familial hypobetalipoproteinemia, FHBL, normotriglyceridemic hypobetalipoproteinemia
Overview
These are a set of diseases caused my mutations in genes involved in triglyceride(TG), cholesterol transport and metabolism. These diseases primarily cause low plasma LDL C and triglyceride levels less than in the 5th percentile of normal population. Clinical manifestations can vary from being completely asymptomatic to multiple features of vitamin deficiencies, and fat malabsorption. Clinical symptoms of vitamin E are seen early in the course of the disease as the amount of vitamin E is parallel to the total lipid level in the body. Failure to diagnose and to initiate timely vitamin supplementation results in the development of neurological symptoms. The mutations causing low LDL levels are widely studied as newer lipid lowering therapies are based on similar mechanisms of these diseases.
Historical Perspective
- In 1960, Salt reported absence of betalipoprotein in the plasma of a patient associated with very low cholesterol levels in the parents. Low cholesterol levels in the parents differentiates familial homozygous hypobetalipoproteinemia from abetalipoproteinemia.[1]
- In 1961, Anderson suggested failure of formation of chylomicron and lipid malabsorption as a cause of severe steatorrhea in children. Patients did not have acanthocytes on the peripheral smear and neuro-ocular symptoms like familial hypobetalipoproteinemia. [2]
- Roy in 1987 and Kane in 1989 described chylomicron retention disease. [3]
- In 2003, the mutation in SAR1B gene was identified by jones.[4]
- Conklin identified the ANGPTL3 gene in 1999 and its function of inhibiting lipoprotein lipase was established in 2013 by Arca.[5][6]
Pathophysiology
Pathogenesis
Hypobetalipoproteinemias are caused by mutations in the genes involved in triglyceride transport and metabolism.
- Cholesterol and triglycerides are insoluble in the plasma and they require a transport protein in the form of apolipoprotein B. These lipoproteins transport cholesterol and trigylcerides in spherical particles with cholesterol esters and triglyceride forming the central core.
- Apolipoprotein B is the major carrier for triglycerides and cholesterol from the intestine and liver to the periphery.
- Apolipoprotein B exits in two forms: apolipoprotein B48 and apolipoprotein B100.
| APOB gene is responsible for the production of Apo B48 in intestine which is critical for the formation and secretion of chylomicrons[7] , and Apo B100 in the liver which is released into circulation as VLDL. | Mutation in the APOB gene affects the translation of mRNA of apolipoprotein B causing familial hypobetalipoproteinemia. The severity of clinical phenotype in familial hypobetalipoproteinemia depends on length of trucated Apo B and zygosity.[8] | ||||||||||||||||||||
| MTP transfers triglycerides from cytsol onto nacent apolipoprotein B in endoplasmic reticulum which is required for assembly and secretion of VLDL and chylomicrons. Mutation in MTP causes abetalipoproteinemia.[9] | In Apo B48 associated chylomicrons, transport of proteins from endoplasmic reticulum to golgi complex is dependent on coat protien complex 2(COP II), secretion-associated, Ras-related GTPase 1B (Sar1b) encoded by the gene SAR1B is a major part of the protein essential for this intra cellular transport.[10] Mutation in Sar1b causes chylomicron retention disease.[4] | ||||||||||||||||||||
| In the periphery by the action of lipoprotein lipase in the endothelium of the capillaries and glycosylphosphatidylinositol-anchored high-density lipoprotein- binding protein 1 (GPIHBP1)[11], a transporter for lipoprotien lipase, triglycerides are hydrolysed to form free fatty acids and glycerol. | |||||||||||||||||||||
| This results in the formation of VLDL remnant(Intermediate density lipoprotein) and chylomicron remnants. The lipases are inhibited by Angiopoietin-like protein 3 (ANGPTL3) thereby decreasing the triglyceride and LDL C.[12][13] | Loss of function mutations or complete absence of ANGPTL3 gene cause familial combined hypolipidemia.[14][15] | ||||||||||||||||||||
| IDL on further removal of triglycerides forms a cholesterol ester rich LDL C. The chylomicron and VLDL remnants removal is apolipoprotein E dependent via the LDL receptors and LDL receptor related protiens.[16] | |||||||||||||||||||||
| LDL C is removed from the circulation by binding to LDL receptors in the liver. The receptor degradation is enhanced by Proprotein convertase subtilisin kexin 9 (PCSK9).[17] | Mutation causing loss of function of the enzyme causes low LDL C levels, and gain of function mutations are associated with familial hypercholesterolemia.[18] | ||||||||||||||||||||
Genetics
The genetic defect, transmission and the result of the mutation in various diseases is described below:
| Homozygous familial
hypobetalipoproteinemia(FHBL) |
Heterozygous familial
hypobetalipoprotienemia |
Chylomicron Retention
Disease |
Familial Combined
Hypolipidemia | |
|---|---|---|---|---|
| Inheritance | Autosomal codominant | Autosomal codominant | Autosomal recessive | Autosomal codominant |
| Defective Gene | APOB gene on chromosome locus 2p23-24 | APOB gene | SAR1B gene on chromosome 5q31 | ANGPTL3 gene on chromosome 1[19] |
| Pathophysiology | Absence of apolipoprotein B results in absent plasma VLDL, triglyceride and LDL C |
|
Intracellular transport of chylomicrons is affected ,resulting in the accumulation of lipids in the cells of the intestine and liver.[20] | Loss of function mutation results in the failure of inhibition of Lipoprotien lipase, leading to low LDL, VLDL and HDL levels. |
- Less common causes of familial hypobetalipoproteinemia are mutations in PCSK9 and ANGPTL3 S17X.[21]
- Mutations in loss of function of PCSK9 do not cause any clinical symptoms but are shown to be associated with decreasing cardiovascular disease risk.[22]
- Mutation in ANGPTL3 S17X causes low LDL C and triglyceride levels along with reduction in plasma glucose level by increasing insulin sensitivity which is secondary to the increased lipoprotien lipase activity.[15]
Causes
The following are the list of causes of primary hypobetalipoproteinemia:
- Abetalipoproteinemia
- Familial hypobetalipoproteinemia
- Chylomicron retention disease
- PCSK9 deficiency
- Familial combined hypolipidemia
Epidemiology and Demographics
The prevalence of these diseases is as follows:[23]
| Prevalence | |
|---|---|
| Abetalipoproteinemia | <1:1,000,000 |
| Familial
Hypobetalipoproteinemia |
1:1000 – 1:3000 |
| Chylomicron Retention
Disease |
Very rare |
| Familial Combined
Hypolipidemia |
Very rare |
| PCSK9 Deficiency | Very rare |
Natural History, complications and Prognosis
| Homozygous Familial Hypobetalipoproteinemia | Heterozygous Familial Hypobetalipoproteinemia | Chylomicron Retention Disease | Familial Combined Hypolipidemia | |
|---|---|---|---|---|
| Disease Course | Steatorrhea early in infancy and progression to neurological symptoms which begin in the 1st or 2nd decade. | Usually benign, few patients may present with steatorrhea. | Early onset of symptoms with diarrhea and failure to thrive. | Benign |
| Complications | Neurologic degeneration, Anemia, Blindness |
|
None | |
| Prognosis |
|
A familial syndrome of longevity has been observed in the benign forms of HBL and many patients live over the age of 85.[27] | Poorly documented evidence on prognosis.[28] | Good |
Diagnosis
History, Symptoms and Physical Examination
Hypobetalipoproteinemias present with varying severity of similar symptoms based on the type of mutation as follows:
| Homozygous Familial
Hypobetalipoproteinemia |
Heterozygous Familial
Hypobetalipoproteinemia |
Chylomicron Retention
Disease |
Familial Combined
Hypolipidemia | |
|---|---|---|---|---|
| Age of Presentation | Infancy | Asymptomatic | 2 months to 1 year | Asymptomatic |
| History and Symptoms |
|
|
|
|
| Physical Examination |
|
Hepatomegaly |
|
Normal Physical Exam |
Laboratory Results
Definitive gold standard for diagnosis is gene sequencing for APOB, MTTP, SAR1B, ANGPTL3 to see the exact mutation. Laboratory findings consistent with the diagnosis of hypobetalipoproteinemias include as follows:
| Homozygous Familial
Hypobetalipoproteinemia |
Heterozygous Familial
Hypobetalipoproteinemia |
Chylomicron Retention
Disease |
Familial Combined
Hypolipidemia | |
|---|---|---|---|---|
| Lipid analysis |
|
| ||
| Other findings |
|
|
|
|
| Abetalipoprotienemia | Familial Homozygous
Hypobetalipoproteinemia |
Familial Heterozygous
Hypobetalipoproteinemia |
PCSK9 deficiency | Chylomicron Retention
Disease |
Familial Combined
Hypolipidemia | |
|---|---|---|---|---|---|---|
| LDL C | ↓↓↓ (0) | ↓↓↓ | ↓ | ↓ | ↓↓ | ↓↓ |
| Apo B | ↓↓↓( 0) | ↓↓↓ | ↓ | N | ↓↓ | N |
| TG | ↓↓↓ | ↓↓↓ | ↓ | ↓ | N | ↓ |
| TC | ↓↓↓ | ↓↓↓ | ↓ | ↓ | ↓↓ | ↓ |
| HDL | ↓↓ | ↓↓ | N | N | ↓↓ | ↓↓ |
| VLDL | ↓↓ | ↓↓ | ↓ | N | ↓↓ | ↓ |
| Apo A1 | ↓↓ | ↓↓ | ↓ | N | ↓↓ | N |
Approach to patient with Low LDL C
| Low LDL C <5th percentile | |||||||||||||||||||||||||||||||
| Rule out secondary causes of low LDL Anemia Criticial illness Chronic inflammation Chronic liver disease Hyperthyroidism Infection Malabsorption Malignancy | |||||||||||||||||||||||||||||||
| Once secondary causes are ruled out consider primary diseases based on analysis of Lipid profile | |||||||||||||||||||||||||||||||
| Normal Triglycerides | Low Triglycerides | ||||||||||||||||||||||||||||||
| Chlyomicron retention disease (Confirm with gene sequencing) | Screen the lipid profile of the patient's parents | ||||||||||||||||||||||||||||||
| Normal Parental Lipid Profile | If Parental Lipid Profile <50% of Normal on: *LDL *Total Cholesterol *Triglycerides | ||||||||||||||||||||||||||||||
| Abetalipoproteinemia (Confirm with gene sequencing) | Familial Homozygous hypobetalipoproteinemia (Confirm with gene sequencing) | ||||||||||||||||||||||||||||||
Treatment
Medical Therapy
- The mainstay of management of familial hypobetalipoproteinemia include early diagnosis and early initiation of low fat diet and fat soluble vitamin supplementation in all symptomatic patients, with yearly follow up to assess the growth and nutritional status, diet compliance, neurological function, lipid panel.
- FHBL heterozygous patients with elevated liver enzyme, regular ultrasound imaging is recommended to monitor for progression of fatty liver to cirrhosis or hepatocellular carcinoma.[40]
Chylomicron Retention Disease Management
- If the patient is diagnosed early in the course of the disease diet modification and oral supplementation of vitamins improved outcomes.[32]
- Low-fat diet
- Vegetable oil enriched in essential fatty acids ± Enriched in medium-chain triglycerides
- Vitamin E (hydrosoluble form): 50 IU/kg/d
- Vitamin A: 15,000 IU/d (adjust according to plasma levels)
- Vitamin D: 800-1200 IU/kg/d or 100,000 IU/2 month if < 5 y old, and 600,000 IU/2 month if > 5 y old
- Vitamin K: 15 mg/week (adjust according to INR and plasma levels)
- If patient is diagnosed late and with neurological disease, combined oral and parental supplementation is recommended:
- Fatty acids-intralipid 20% 2g/kg/month
- Vitamin E 4 to 6 mg/kg/month
- Vitamin A 500 IU/kg/month once a month is recommended
Follow up
- Annual follow up to 10 years to assess the growth and nutritional status, diet compliance, neurological function, lipid profile.
- Every 3 year follow up to check bone mineral density, liver function with ultrasound, ophthalmologic exam for fundus, color vision, visual evoked potentials and electroretinography after the age of 10years.
- Echocardiography in adulthood.
Surgical Therapy
- No surgical options are available.
Prevention
Primary Prevention
- As the set of the diseases are rare there are no primary preventive measures.
Secondary Prevention
- Regular follow up to look for complications and strict adherence to therapy has shown to prevent progression of the disease.
References
- ↑ SALT HB, WOLFF OH, LLOYD JK, FOSBROOKE AS, CAMERON AH, HUBBLE DV (1960). "On having no beta-lipoprotein. A syndrome comprising a-beta-lipoproteinaemia, acanthocytosis, and steatorrhoea". Lancet. 2 (7146): 325–9. PMID 13745738.
- ↑ ANDERSON CM, TOWNLEY RR, JOHANSEN P (1961). "Unusual causes of steatorrhoea in infancy and childhood". Med J Aust. 48(2): 617–22. PMID 13861205.
- ↑ Roy CC, Levy E, Green PH, Sniderman A, Letarte J, Buts JP; et al. (1987). "Malabsorption, hypocholesterolemia, and fat-filled enterocytes with increased intestinal apoprotein B. Chylomicron retention disease". Gastroenterology. 92 (2): 390–9. PMID 3792776.
- ↑ 4.0 4.1 Jones B, Jones EL, Bonney SA, Patel HN, Mensenkamp AR, Eichenbaum-Voline S; et al. (2003). "Mutations in a Sar1 GTPase of COPII vesicles are associated with lipid absorption disorders". Nat Genet. 34 (1): 29–31. doi:10.1038/ng1145. PMID 12692552.
- ↑ Arca M, Minicocci I, Maranghi M (2013). "The angiopoietin-like protein 3: a hepatokine with expanding role in metabolism". Curr Opin Lipidol. 24 (4): 313–20. doi:10.1097/MOL.0b013e3283630cf0. PMID 23839332.
- ↑ Conklin D, Gilbertson D, Taft DW, Maurer MF, Whitmore TE, Smith DL; et al. (1999). "Identification of a mammalian angiopoietin-related protein expressed specifically in liver". Genomics. 62 (3): 477–82. doi:10.1006/geno.1999.6041. PMID 10644446.
- ↑ Dash S, Xiao C, Morgantini C, Lewis GF (2015). "New Insights into the Regulation of Chylomicron Production". Annu Rev Nutr. 35: 265–94. doi:10.1146/annurev-nutr-071714-034338. PMID 25974693.
- ↑ Di Leo E, Eminoglu T, Magnolo L, Bolkent MG, Tümer L, Okur I; et al. (2015). "The Janus-faced manifestations of homozygous familial hypobetalipoproteinemia due to apolipoprotein B truncations". J Clin Lipidol. 9 (3): 400–5. doi:10.1016/j.jacl.2015.01.005. PMID 26073401.
- ↑ Berriot-Varoqueaux N, Aggerbeck LP, Samson-Bouma M, Wetterau JR (2000). "The role of the microsomal triglygeride transfer protein in abetalipoproteinemia". Annu Rev Nutr. 20: 663–97. doi:10.1146/annurev.nutr.20.1.663. PMID 10940349.
- ↑ Shoulders CC, Stephens DJ, Jones B (2004). "The intracellular transport of chylomicrons requires the small GTPase, Sar1b". Curr Opin Lipidol. 15 (2): 191–7. PMID 15017362.
- ↑ Young SG, Davies BS, Voss CV, Gin P, Weinstein MM, Tontonoz P; et al. (2011). "GPIHBP1, an endothelial cell transporter for lipoprotein lipase". J Lipid Res. 52 (11): 1869–84. doi:10.1194/jlr.R018689. PMC 3196223. PMID 21844202.
- ↑ Shan L, Yu XC, Liu Z, Hu Y, Sturgis LT, Miranda ML; et al. (2009). "The angiopoietin-like proteins ANGPTL3 and ANGPTL4 inhibit lipoprotein lipase activity through distinct mechanisms". J Biol Chem. 284 (3): 1419–24. doi:10.1074/jbc.M808477200. PMC 3769808. PMID 19028676.
- ↑ Yoshida K, Shimizugawa T, Ono M, Furukawa H (2002). "Angiopoietin-like protein 4 is a potent hyperlipidemia-inducing factor in mice and inhibitor of lipoprotein lipase". J Lipid Res. 43 (11): 1770–2. PMID 12401877.
- ↑ Romeo S, Yin W, Kozlitina J, Pennacchio LA, Boerwinkle E, Hobbs HH; et al. (2009). "Rare loss-of-function mutations in ANGPTL family members contribute to plasma triglyceride levels in humans". J Clin Invest. 119 (1): 70–9. doi:10.1172/JCI37118. PMC 2613476. PMID 19075393.
- ↑ 15.0 15.1 Robciuc MR, Maranghi M, Lahikainen A, Rader D, Bensadoun A, Öörni K; et al. (2013). "Angptl3 deficiency is associated with increased insulin sensitivity, lipoprotein lipase activity, and decreased serum free fatty acids". Arterioscler Thromb Vasc Biol. 33 (7): 1706–13. doi:10.1161/ATVBAHA.113.301397. PMID 23661675.
- ↑ Lillis AP, Van Duyn LB, Murphy-Ullrich JE, Strickland DK (2008). "LDL receptor-related protein 1: unique tissue-specific functions revealed by selective gene knockout studies". Physiol Rev. 88 (3): 887–918. doi:10.1152/physrev.00033.2007. PMC 2744109. PMID 18626063.
- ↑ Garvie CW, Fraley CV, Elowe NH, Culyba EK, Lemke CT, Hubbard BK; et al. (2016). "Point mutations at the catalytic site of PCSK9 inhibit folding, autoprocessing, and interaction with the LDL receptor". Protein Sci. 25 (11): 2018–2027. doi:10.1002/pro.3019. PMC 5079255. PMID 27534510.
- ↑ Marais AD, Kim JB, Wasserman SM, Lambert G (2015). "PCSK9 inhibition in LDL cholesterol reduction: genetics and therapeutic implications of very low plasma lipoprotein levels". Pharmacol Ther. 145: 58–66. doi:10.1016/j.pharmthera.2014.07.004. PMID 25046268.
- ↑ Fazio S, Sidoli A, Vivenzio A, Maietta A, Giampaoli S, Menotti A; et al. (1991). "A form of familial hypobetalipoproteinaemia not due to a mutation in the apolipoprotein B gene". J Intern Med. 229 (1): 41–7. PMID 1995762.
- ↑ Charcosset M, Sassolas A, Peretti N, Roy CC, Deslandres C, Sinnett D; et al. (2008). "Anderson or chylomicron retention disease: molecular impact of five mutations in the SAR1B gene on the structure and the functionality of Sar1b protein". Mol Genet Metab. 93 (1): 74–84. doi:10.1016/j.ymgme.2007.08.120. PMID 17945526.
- ↑ 21.0 21.1 21.2 Minicocci I, Montali A, Robciuc MR, Quagliarini F, Censi V, Labbadia G; et al. (2012). "Mutations in the ANGPTL3 gene and familial combined hypolipidemia: a clinical and biochemical characterization". J Clin Endocrinol Metab. 97 (7): E1266–75. doi:10.1210/jc.2012-1298. PMID 22659251.
- ↑ Cohen JC, Boerwinkle E, Mosley TH, Hobbs HH (2006). "Sequence variations in PCSK9, low LDL, and protection against coronary heart disease". N Engl J Med. 354 (12): 1264–72. doi:10.1056/NEJMoa054013. PMID 16554528.
- ↑ De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, Shapiro MD. PMID 26561704. Missing or empty
|title=(help) - ↑ Cefalù AB, Pirruccello JP, Noto D, Gabriel S, Valenti V, Gupta N; et al. (2013). "A novel APOB mutation identified by exome sequencing cosegregates with steatosis, liver cancer, and hypocholesterolemia". Arterioscler Thromb Vasc Biol. 33 (8): 2021–5. doi:10.1161/ATVBAHA.112.301101. PMC 3870266. PMID 23723369.
- ↑ Lacaille F, Bratos M, Bouma ME, Jos J, Schmitz J, Rey J (1989). "[Anderson's disease. Clinical and morphologic study of 7 cases]". Arch Fr Pediatr. 46 (7): 491–8. PMID 2596948.
- ↑ Zamel R, Khan R, Pollex RL, Hegele RA (2008). "Abetalipoproteinemia: two case reports and literature review". Orphanet J Rare Dis. 3: 19. doi:10.1186/1750-1172-3-19. PMC 2467409. PMID 18611256.
- ↑ "Orphanet: Hypobetalipoproteinemia".
- ↑ "Orphanet: Chylomicron retention disease".
- ↑ Tarugi P, Lonardo A, Gabelli C, Sala F, Ballarini G, Cortella I; et al. (2001). "Phenotypic expression of familial hypobetalipoproteinemia in three kindreds with mutations of apolipoprotein B gene". J Lipid Res. 42 (10): 1552–61. PMID 11590210.
- ↑ Tanoli T, Yue P, Yablonskiy D, Schonfeld G (2004). "Fatty liver in familial hypobetalipoproteinemia: roles of the APOB defects, intra-abdominal adipose tissue, and insulin sensitivity". J Lipid Res. 45 (5): 941–7. doi:10.1194/jlr.M300508-JLR200. PMID 14967820.
- ↑ Peretti N, Roy CC, Sassolas A, Deslandres C, Drouin E, Rasquin A; et al. (2009). "Chylomicron retention disease: a long term study of two cohorts". Mol Genet Metab. 97 (2): 136–42. doi:10.1016/j.ymgme.2009.02.003. PMID 19285442.
- ↑ 32.0 32.1 Peretti N, Sassolas A, Roy CC, Deslandres C, Charcosset M, Castagnetti J; et al. (2010). "Guidelines for the diagnosis and management of chylomicron retention disease based on a review of the literature and the experience of two centers". Orphanet J Rare Dis. 5: 24. doi:10.1186/1750-1172-5-24. PMC 2956717. PMID 20920215.
- ↑ Lee J, Hegele RA (2014). "Abetalipoproteinemia and homozygous hypobetalipoproteinemia: a framework for diagnosis and management". J Inherit Metab Dis. 37 (3): 333–9. doi:10.1007/s10545-013-9665-4. PMID 24288038.
- ↑ Welty FK, Lichtenstein AH, Barrett PH, Dolnikowski GG, Ordovas JM, Schaefer EJ (1997). "Decreased production and increased catabolism of apolipoprotein B-100 in apolipoprotein B-67/B-100 heterozygotes". Arterioscler Thromb Vasc Biol. 17 (5): 881–8. PMID 9157951.
- ↑ Elias N, Patterson BW, Schonfeld G (1999). "Decreased production rates of VLDL triglycerides and ApoB-100 in subjects heterozygous for familial hypobetalipoproteinemia". Arterioscler Thromb Vasc Biol. 19 (11): 2714–21. PMID 10559016.
- ↑ Georges A, Bonneau J, Bonnefont-Rousselot D, Champigneulle J, Rabès JP, Abifadel M; et al. (2011). "Molecular analysis and intestinal expression of SAR1 genes and proteins in Anderson's disease (Chylomicron retention disease)". Orphanet J Rare Dis. 6: 1. doi:10.1186/1750-1172-6-1. PMC 3029219. PMID 21235735.
- ↑ Musunuru K, Pirruccello JP, Do R, Peloso GM, Guiducci C, Sougnez C; et al. (2010). "Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia". N Engl J Med. 363 (23): 2220–7. doi:10.1056/NEJMoa1002926. PMC 3008575. PMID 20942659.
- ↑ Dannoura AH, Berriot-Varoqueaux N, Amati P, Abadie V, Verthier N, Schmitz J; et al. (1999). "Anderson's disease: exclusion of apolipoprotein and intracellular lipid transport genes". Arterioscler Thromb Vasc Biol. 19 (10): 2494–508. PMID 10521380.
- ↑ Gusarova V, Brodsky JL, Fisher EA (2003). "Apolipoprotein B100 exit from the endoplasmic reticulum (ER) is COPII-dependent, and its lipidation to very low density lipoprotein occurs post-ER". J Biol Chem. 278 (48): 48051–8. doi:10.1074/jbc.M306898200. PMID 12960170.
- ↑ Welty FK (2014). "Hypobetalipoproteinemia and abetalipoproteinemia". Curr Opin Lipidol. 25 (3): 161–8. doi:10.1097/MOL.0000000000000072. PMC 4465983. PMID 24751931.