Case#: (Biallelic affected sib) MM, female, 10 years (at age of blood draw) CaseHPOs: HP:0002024, HP:0002013, HP:0002570, HP:0002495, HP:0001284 (malabsorption, vomiting, steatorrhea, , impaired vibratory sensation, absent deep tendon reflexes)

CaseHPOsFreeText:: Diagnosed with FHBL at age 10 years; neurological symptoms stabilized on Vitamin E treatment. CasePreviousTesting: Total cholesterol 24mg/dl, no detectable triglyceride ot LDL-cholesterol, 21 mg/dl HDL cholesterol. Variant: Compound heterozygous: Variant 1: NM_000384.2:c.537+1G>T (paternal) Variant 2: NM_000384.2:c.1315C>T (p.Arg439Ter) (maternal)

ClinVar: N/A

CAID: Variant 1: CA345962364 ; Variant 2: CA052017

gnomAD: Variant 1: Not in gnomAD; Variant 2: 0.00002640 (Euro non-Finnish)

Case#: (Heterozygous sib) GM (II-5), male, 14 years (at blood draw), sib of proband CaseHPOs: HP:0003563 CaseHPOsFreeText: Asymptomatic CasePreviousTesting: Total cholesterol 97 mg/dl, triglyceride 59 mg/dl, LDL-cholesterol 29 mg/dl (<5th percentile), HDL cholesterol 56 mg/dl. ** Variant:** Heterozygous for NM_000384.2:c.537+1G>T (paternal)

CAID: CA345962364

Case#: (Heterozygous sib) JM (II-6), male, 7 years (at blood draw), sib of proband CaseHPOs: HP:0003563 CaseHPOsFreeText: Asymptomatic CasePreviousTesting: Total cholesterol 113 mg/dl, triglyceride 60 mg/dl, LDL-cholesterol 16 mg/dl (<5th percentile), HDL cholesterol 87 mg/dl) ** Variant:** Heterozygous for NM_000384.2:c.1315C>T (p.Arg439Ter) (maternal)

CAID: CA052017

Case#: (Biallelic affected sib) MM, female, 10 years (at age of blood draw) CaseHPOs: HP:0002024, HP:0002013, HP:0002570, HP:0002495, HP:0001284 (malabsorption, vomiting, steatorrhea, , impaired vibratory sensation, absent deep tendon reflexes)

CaseHPOsFreeText:: Diagnosed with FHBL at age 10 years; neurological symptoms stabilized on Vitamin E treatment. CasePreviousTesting: Total cholesterol 24mg/dl, no detectable triglyceride ot LDL-cholesterol, 21 mg/dl HDL cholesterol. Variant: Compound heterozygous: Variant 1: NM_000384.2:c.537+1G>T (paternal) Variant 2: NM_000384.2:c.1315C>T (p.Arg439Ter) (maternal)

ClinVar: N/A

CAID: Variant 1: CA345962364 ; Variant 2: CA052017

gnomAD: Variant 1: Not in gnomAD; Variant 2: 0.00002640 (Euro non-Finnish)

Case#: (Heterozygous sib) GM (II-5), male, 14 years (at blood draw), sib of proband CaseHPOs: HP:0003563 CaseHPOsFreeText: Asymptomatic CasePreviousTesting: Total cholesterol 97 mg/dl, triglyceride 59 mg/dl, LDL-cholesterol 29 mg/dl (<5th percentile), HDL cholesterol 56 mg/dl. ** Variant:** Heterozygous for NM_000384.2:c.537+1G>T (paternal)

CAID: CA345962364

Case#: (Heterozygous sib) JM (II-6), male, 7 years (at blood draw), sib of proband CaseHPOs: HP:0003563 CaseHPOsFreeText: Asymptomatic CasePreviousTesting: Total cholesterol 113 mg/dl, triglyceride 60 mg/dl, LDL-cholesterol 16 mg/dl (<5th percentile), HDL cholesterol 87 mg/dl) ** Variant:** Heterozygous for NM_000384.2:c.1315C>T (p.Arg439Ter) (maternal)

CAID: CA052017

Case#: Family IV-II:1, 58 years old, male

CaseHPOs: HP:0003563 (decreased LDL cholesterol)

CaseHPOsFreeText: Type 2 diabetes and severe non-alcoholic steatohepatitis (NASH) with pre-cirrhotic lesions and well differentiated hepatocellular carcinoma (HCC) diagnosed on liver biopsy

CasePreviousTesting: TC 120 mg/dl, LDL-C 50 mg/dl, HDL-C 55 mg/dl, TG 89 mg/dl (see additional values in Table 1) ApoB100 19 mg/dl (normal range 49-88) (Supplemental data)

Variant: NM_000384.2:c.11153C>G (p.Ser3718Ter), heterozygous

CAID: CA345983128

gnomAD: Absent

Variant evidence: APOB- p.Ser3718* mutations lead to the formation of expected truncated ApoB forms (ApoB-81) of 418 kDa (Fig. 1C, right panel) that can be secreted by the liver and lipidated.

CaseAddInfo: Son of Family IV-I:1

Case#: Family II-II:1, 41 year old, male

CaseHPOs: HP:0003563, HP:0001397 (decreased LDL cholesterol, hepatic steatosis)

CaseHPOsFreeText: Prediabetes

CasePreviousTesting: TC 113 mg/dl, LDL-C 38 mg/dl, HDL-C 69 mg/dl, TG 32 mg/dl (see additional values in Table 1). ApoB100 22 mg/dl (normal range 49-88) (Supplemental data). Normal ranges- total cholesterol (TC) 92-234 mg/dl, LDL-cholesterol (LDL-C) 136-174 mg/dl, HDL-cholesterol (HDL-C) 60-95 mg/dl), triglyceride (TG) 35-132 mg/dl.

CaseGenotypingMethod: Next generation sequencing panel for high-throughput sequencing of the coding regions of genes previously linked to FHBL (APOB, PCSK9, ANGPTL3) and ABL (MTTP, SAR1B). Sanger sequencing was performed for validation.

Variant: NM_000384.2:c.7711C>T (p.Gln2571Ter), heterozygous

CAID: CA43502110

gnomAD: Absent

Family#: Family III

PresentingHPOs: HP:0003563 (decreased LDL cholesterol)

PresentingHPOsFreeText: FHBL diagnosis was based on low levels of fasting calculated plasma LDL-C < 50 mg/dL without lipid lowering therapy. Affected relatives were diagnosed by LDL-C < 80 mg/dL and/or ApoB <50 mg/dL.

FamilyMethod1: Next generation sequencing

FamilyGenotypingMethod: Next generation sequencing panel for high-throughput sequencing of the coding regions of genes previously linked to FHBL (APOB, PCSK9, ANGPTL3) and ABL (MTTP, SAR1B). Sanger sequencing was performed for validation and co-segregation analysis among family members.

ProbandLabel: Family III-II:2

Variant: NM_000384.2:c.8799C>A (p.Cys2933*), heterozygous

CAID: CA345993342

gnomAD: Absent

Case#: Family IV-II:1, 58 years old, male

CaseHPOs: HP:0003563 (decreased LDL cholesterol)

CaseHPOsFreeText: Type 2 diabetes and severe non-alcoholic steatohepatitis (NASH) with pre-cirrhotic lesions and well differentiated hepatocellular carcinoma (HCC) diagnosed on liver biopsy

CasePreviousTesting: TC 120 mg/dl, LDL-C 50 mg/dl, HDL-C 55 mg/dl, TG 89 mg/dl (see additional values in Table 1) ApoB100 19 mg/dl (normal range 49-88) (Supplemental data)

Variant: NM_000384.2:c.11153C>G (p.Ser3718Ter), heterozygous

CAID: CA345983128

gnomAD: Absent

Variant evidence: APOB- p.Ser3718* mutations lead to the formation of expected truncated ApoB forms (ApoB-81) of 418 kDa (Fig. 1C, right panel) that can be secreted by the liver and lipidated.

CaseAddInfo: Son of Family IV-I:1

Case#: Family II-II:1, 41 year old, male

CaseHPOs: HP:0003563, HP:0001397 (decreased LDL cholesterol, hepatic steatosis)

CaseHPOsFreeText: Prediabetes

CasePreviousTesting: TC 113 mg/dl, LDL-C 38 mg/dl, HDL-C 69 mg/dl, TG 32 mg/dl (see additional values in Table 1). ApoB100 22 mg/dl (normal range 49-88) (Supplemental data). Normal ranges- total cholesterol (TC) 92-234 mg/dl, LDL-cholesterol (LDL-C) 136-174 mg/dl, HDL-cholesterol (HDL-C) 60-95 mg/dl), triglyceride (TG) 35-132 mg/dl.

CaseGenotypingMethod: Next generation sequencing panel for high-throughput sequencing of the coding regions of genes previously linked to FHBL (APOB, PCSK9, ANGPTL3) and ABL (MTTP, SAR1B). Sanger sequencing was performed for validation.

Variant: NM_000384.2:c.7711C>T (p.Gln2571Ter), heterozygous

CAID: CA43502110

gnomAD: Absent

Family#: Family III

PresentingHPOs: HP:0003563 (decreased LDL cholesterol)

PresentingHPOsFreeText: FHBL diagnosis was based on low levels of fasting calculated plasma LDL-C < 50 mg/dL without lipid lowering therapy. Affected relatives were diagnosed by LDL-C < 80 mg/dL and/or ApoB <50 mg/dL.

FamilyMethod1: Next generation sequencing

FamilyGenotypingMethod: Next generation sequencing panel for high-throughput sequencing of the coding regions of genes previously linked to FHBL (APOB, PCSK9, ANGPTL3) and ABL (MTTP, SAR1B). Sanger sequencing was performed for validation and co-segregation analysis among family members.

ProbandLabel: Family III-II:2

Variant: NM_000384.2:c.8799C>A (p.Cys2933*), heterozygous

CAID: CA345993342

gnomAD: Absent

Case#: DD, 21 years, female

CasePresentingHPOsFreeText: apo-B plasma levels <2% of normal and > 50% acanthocytes; total cholesterol 1.27 (normal range 4-6.5 mmol/l), triglyceride 0.3 (normal range 0.3-1.8 mmol/l), apoB 1.5 (normal range 60-140 mg/dl), “virtually no LDL”; diagnosed with “homozygous” (i.e. biallelic) familial hypobetalipoproteinemia because both parents (AD and PD) had total plasma cholesterol and apo-B levels below the 5th percentile. Designated as the proband in the paper. However, only one variant identified (note that full gene sequencing was not performed”), and therefore PD (mother of DD) was added as the proband here as she is heterozygous for the variant and has lipid levels consistent with this.

PresentingNotHPOsFreeText: No neurological or retinal disease.

CasePreviousTesting: On Western blot a truncated species of apo-B was found in the VLDL/chylomicron fraction (fig. 3), predicted based on size to be at the 5’ end of exon 26. The protein was absent from the HDL or infranate fractions.

CaseGenotypingMethod: Genomic DNA was digested with a panel of restriction enzymes and Southern blotted. Based on truncated apoB size (detected on Western blot), the variant was predicted to be at the end of exon 26. Approximately 1.7 kb of genomic DNA including exon 26 was amplified by the polymerase chain reaction (PCR), cloned into M13, and sequenced.

Variant: Variant 1: NM_000384.3(APOB):c.5463del (p.His1822fs)

ClinVar: Variant 1 17882 Variant 2 – Not identified (not that full gene sequencing was not performed)

gnomAD: Variant 1 - 0.000008797 (European non-Finnish) Variant 2 – Not identified ((not that full gene sequencing was not performed)

CaseAddInfo: PD (mother) had the truncated apo-B (apo-B39) in both her VLDL and LDL lipoprotein fractions, but it was present at reduced levels compared to the apo-B100 arising from her one normal apo-B allele (fig. 3). Only apo-B100 and apo-B48 were detected in the father, AD.

Case#: DD, 21 years, female

CasePresentingHPOsFreeText: apo-B plasma levels <2% of normal and > 50% acanthocytes; total cholesterol 1.27 (normal range 4-6.5 mmol/l), triglyceride 0.3 (normal range 0.3-1.8 mmol/l), apoB 1.5 (normal range 60-140 mg/dl), “virtually no LDL”; diagnosed with “homozygous” (i.e. biallelic) familial hypobetalipoproteinemia because both parents (AD and PD) had total plasma cholesterol and apo-B levels below the 5th percentile. Designated as the proband in the paper. However, only one variant identified (note that full gene sequencing was not performed”), and therefore PD (mother of DD) was added as the proband here as she is heterozygous for the variant and has lipid levels consistent with this.

PresentingNotHPOsFreeText: No neurological or retinal disease.

CasePreviousTesting: On Western blot a truncated species of apo-B was found in the VLDL/chylomicron fraction (fig. 3), predicted based on size to be at the 5’ end of exon 26. The protein was absent from the HDL or infranate fractions.

CaseGenotypingMethod: Genomic DNA was digested with a panel of restriction enzymes and Southern blotted. Based on truncated apoB size (detected on Western blot), the variant was predicted to be at the end of exon 26. Approximately 1.7 kb of genomic DNA including exon 26 was amplified by the polymerase chain reaction (PCR), cloned into M13, and sequenced.

Variant: Variant 1: NM_000384.3(APOB):c.5463del (p.His1822fs)

ClinVar: Variant 1 17882 Variant 2 – Not identified (not that full gene sequencing was not performed)

gnomAD: Variant 1 - 0.000008797 (European non-Finnish) Variant 2 – Not identified ((not that full gene sequencing was not performed)

CaseAddInfo: PD (mother) had the truncated apo-B (apo-B39) in both her VLDL and LDL lipoprotein fractions, but it was present at reduced levels compared to the apo-B100 arising from her one normal apo-B allele (fig. 3). Only apo-B100 and apo-B48 were detected in the father, AD.