In our recent Lean Mass Hyper-Responder (LMHR) case report, we performed extensive genetic testing on the subject, LM, and activity sought input from clinicians and geneticists outside the research team. Because of the broad nature of our testing and absence of notable findings, as determined by expert consultants, and manuscript length limitations, we stated in our publication: “Whole exome sequencing performed by Veritas Genetics, and independent dyslipidemia and ASCVD genetic risk testing by GB Healthwatch, revealed no pathogenic or likely pathogenic variants that could account for LM’s phenotype.”

As part of our continued commitment to open and transparent science, we are happy to disclose what results we can. But before doing so below, it is important to highlight two caveats:

1. While a whole exome sequence was ordered, it would obviously be inappropriate to release the entire Variant Call Format file. This represents the subject’s actual genetic code and to disclose the code would be a serious infringement on the subject’s privacy, and one that could potentially be used to his disadvantage in future. Thus, calls for access to the complete raw exome sequence by individuals not part of the patient’s care team will not be entertained.

2. Given the scope of the genetic testing performed, it is inevitable variants will be found. Every person carries risk variants. The important question is whether variant can explain the clinical and metabolic (LMHR) phenotype at hand. By way of example, a risk variant for elevated triglycerides is not clinically relevant if the subject’s triglycerides are 40 mg/dl.

Moving onto the genetic findings: In addition to the exome sequence with professional geneticist interpretation, we also ordered a Dyslipidemia and ASCVD comprehensive risk panel — the same one that is being ordered for the LMHR prospective trial (recruitment underway) and that includes the following targets: LDLR, APOB, PCSK9, LDLRAP1, LPL, CETP, LCAT, LIPC, LIPE, LIPG, LPA, PPARG, STAP1, ABCA5, ABCA6, ABCG5, APOC2, LMF1, GBIHBP1, CREB3L3, GCKR, SCARB1, ABCA1, APOA1, APOA5, BHMT, CBS, MTHR, MTR, MTRR, PEMT, SHMT1, GUCY1A1, ITGB3, MEF2A, NOS3, PLA2G7.

As shown below, and consistent with the published text, testing by GB Healthwatch, “revealed no [0] pathogenic or likely pathogenic variants that could account for LM’s phenotype.”

In terms of variants not classed as pathogenic/likely pathogenic, there were no variants in genes classically associated with familial hypercholesterolemia at levels seen in this patient: LDLR, APOB, LDLRAP1, or PCSK9. Overall, six potentially notable variants were identified, as follows:

Pathogenic and Likely Pathogenic Variants (0) and Variants of Uncertain Significance (VUS) and High-Risk Variants, provided by GB Healthwatch

• ABCG8 521G>A, heterozygous VUS for sitosterolemia. Sitosterolemia is a condition in which plant sterols and other sterols can be hyperabsorbed. Given that this is a hyperabsorption disorder, the mainstay of therapy is dietary restriction of both cholesterol and plant sterols. Of note: (i) testing performed on LM on October 20, 2021 confirmed normal campesterol levels at 8.2 mg/L. (ii) Most importantly, a hyperabsorption phenotype is inconsistent with the clinical presentation. As reported in the manuscript, when hyperabsorption was considered in the case of LM, “LM was recommended to reduce dietary cholesterol intake, eliminating liver, shellfish, and egg yolks from his diet (in substitution for lean chicken, fish, and egg whites). One month later, in September 2020, his LDL-C was remeasured at 545 mg/dl (HDL-C 94 mg/dl, TG 58 mg/dl).” Thus, LM’s dietary cholesterol intake was lowest when his LDL-C was at its peak, and hyperabsorption cannot account for his LDL-C phenotype on a carbohydrate restricted diet.
• APOA5 3’UTR, heterozygous variant and risk factor for hypertriglyceridemia. The patient’s triglycerides ranged from 39 – 58 mg/dL. Again, the risk variant is inconsistent with the metabolic phenotype.
• CBS 133C>T missense heterozygous VUS for homocystinuria. LM has no clinical signs of this disorder and homocysteine last measured January 3^rd 2020 normal at 9.7 umol/L.
• CDKN2B-AS1 22124478A>G homozygous variant that codes for an altered form of a long non-coding RNA that is associated with increased risk of myocardial infarction in some studies. While this variant does associate with increased risk of myocardial infarction, the increased risk does not appear to be driven by alternations in blood lipids, including LDL-C and there is no reason to believe it contributed to the patient’s phenotype on a carbohydrate restricted diet.
• LPL 953A>G heterozygous variant that has been linked to increased risk for hypertriglyceridemia. As noted, the patient’s triglycerides ranged from 39 – 58 mg/dL. Again, the risk variant is inconsistent with the metabolic phenotype.
• SLC22A1 1022C>T heterozygous variant and risk factor for elevated Lp(a). As stated in the report, Lp(a) was high in the subject prior to adopting a ketogenic diet and is elevated in the patient’s father. This allele is presumably paternally inherited and there is no reason to believe it contributed to the patient’s LMHR phenotype.

In addition to the targeted genetic risk panel, a 144-page report corresponding to the exome sequence also revealed no known pathogenic or likely pathogenic variants for heart disease. Under “important” risk variants, the patient was noted to be a carrier for HFE 845G>A for hereditary hemochromatosis, MEFV 442G>C for Familial Mediterranean Fever, and MMP2 524G>A for multicentric osteolysis, nodulosis, and anthropopathy. These are all autosomal recessive conditions, and the patient does not present with signs or symptoms of any of these disorders. Homozygosity for lactose intolerance was also noted.

Under “noteworthy” variants, 7 variants were identified for cancer risk, 3 for clotting disorders, 4 for neurological disorders, 4 for other organ health, and only one for cardiovascular disease. This variant was in KCNE1 with classification of “no known risk” (VUS), for long QT syndrome, as detailed below. Taken together, the exome sequence revealed no notable findings that could explain the patient’s LMHR phenotype.

Thus, these genetic data provide no means by which to explain the patients presentation, defined by a shift from normal LDL-C of 95 mg/dl while on a mixed macronutrient diet to an LMHR triad of LDL-C 393 – 545 mg/dl, HDL-C ~115 mg/dl, triglycerides ~40 mg/dl. In fact, several of the identified variants were associated with increased risk for hypertriglyceridemia, which is in obvious contrast to the patient’s presentation.

In summary, our interpretation is that, while one cannot rule out genetic contribution or modification, the evidence at hand is most consistent with the hypothesis that the LMHR phenotype is driven by non-genetic factors including leanness and dietary macronutrient composition. More details on the Lipid Energy Model will be forthcoming shortly.