Written informed consent for the genetic analysis and publication of associated data was obtained from all the investigated family members.
At admission, the proband (II.5) was a 48 year-old male who was referred for cardiac stress test and dyslipidemia management to the cardiology department of hospital. The proband had elevated LDL-C levels (263mg/dL; 6.8mmol/L) and was on lipid lowering therapies (LLTs) for 8 years. He eventually underwent percutaneous transluminal coronary angioplasty (PTCA) in 2019. Cascade screening of family members revealed that the proband’s mother (I.2) and all his three siblings (II.2, II.4, II.7) had an elevated lipid levels (LDL-C > 155mg/dL; 4.01mmol/L) at the time of their diagnosis and were managed by LLTs (Fig. 1). The proband’s father (I.1) was apparently healthy with no symptoms of dyslipidemia or CAD until his death at the age of 78 years, however the proband’s two siblings (II.2, II.4), succumbed to myocardial infarction at the age of 50 and 45 years respectively.
The second generation in the family i.e., II.2, II.4, II.5 were married to a healthy individuals i.e. II.1, II.3, II.6 respectively, with no personal/family history of hypercholesterolemia/CAD (Fig. 2). However, proband’s younger brother, II.7 entered into a consanguineous marriage with II.8 (maternal uncle’s daughter) who too had elevated LDL-C levels (285mg/dl; 7.37mmol/L). Their children, III.5 & III.6 were diagnosed with dyslipidemia at their age of 3yrs & 9yrs respectively and were treated with aggressive LLTs since then. III.5 had developed cutaneous and tendinous xanthomas at an early age and died because of premature CAD at the age of 15. Other children from the third generation i.e. III.1, III.3 and III.4 are also dyslipidemic with the former i.e. III.1 underwent PTCA at the age of 25 years. All of them are currently on LLTs. Information for III.2 was not available. Demographics of family members are shown in Table 1 (detailed information in Supplementary).
Table 1: Demographic details of the Indian Family members:
Generations
|
Age
(yrs)
|
Sex
|
Clinically Affected
|
Lipid Lowering therapies (LLT)
|
LDLR c.743_744delinsAA mutation (heterozygous)
|
DLNC - Category (Score)
|
I
|
II
|
III
|
|
|
|
|
|
|
I.1*
|
|
|
78
|
M
|
No
|
No
|
*
|
Unlikely (1)
|
I.2*
|
|
|
80
|
F
|
Yes
|
Yes
|
Present
|
Definite (12)
|
|
II.1
|
|
65
|
M
|
No
|
No
|
Not tested
|
Possible (4)
|
|
II.2*
|
|
50
|
F
|
Yes
|
Yes
|
*
|
Possible (5)
|
|
|
III.1
|
25
|
M
|
Yes (PTCA)
|
Yes
|
Not tested
|
Probable (7)
|
|
|
III.2
|
30
|
F
|
Not known
|
Not known
|
Not tested
|
Possible (3)
|
|
II.3
|
|
50
|
F
|
No
|
No
|
Not tested
|
Unlikely(1)
|
|
II.4*
|
|
45
|
M
|
Yes
|
Yes
|
*
|
Possible (5)
|
|
|
III.3
|
25
|
M
|
Yes
|
Yes
|
Not tested
|
Probable (8)
|
|
II.5
(Proband)
|
|
48
|
M
|
Yes (PTCA)
|
Yes
|
Present
|
Definite (16)
|
|
II.6
|
|
45
|
F
|
No
|
No
|
Absent
|
Unlikely (1)
|
|
|
III.4
|
15
|
M
|
Yes
|
Yes
|
Present
|
Definite (14)
|
|
II.7
|
|
45
|
M
|
Yes
|
Yes
|
Present
|
Definite (12)
|
|
II.8
|
|
40
|
F
|
Yes
|
Yes
|
Present
|
Definite (12)
|
|
|
III.5*
|
15
|
M
|
Yes
|
Yes
|
*
|
Definite (15)
|
|
|
III.6
|
9
|
F
|
Yes
|
Yes
|
Present
|
Definite (16)
|
*Deceased, PTCA - Percutaneous transluminal coronary angioplasty, DLNC-Dutch Lipid Network Criteria; Score -Definite ≥8, Probable 6-7, Possible, 3-5 Unlikely <3 (according to DLNC), M - Male, F - Female
Genetic Analysis:
EDTA blood samples were collected from the investigated family members followed by DNA extraction [10]. Initially genetic analysis was not performed on the family. However, the proband’s niece (III.6) who as per DLNC criteria was classified as Definite FH and was included in an ongoing Indian Council of Medical Research funded project on FH. The project aimed to determine the mutation spectrum of Indian Definite FH patients by Whole exome sequencing. Upon analysis, it was revealed that the proband’s niece, III.6 harbored a novel nonsense mutation in exon 5 of LDLR gene (Supplementary figure). According to the ACMG-AMP guidelines and HGVS nomenclature, this novel mutation found was described as c.743_744delinsAA (NP_00518.1:p.Cys248Ter;C248*) (Fig. 3). The novel mutation has been submitted in the ClinVar database [Accession number: SCV004244681]. Furthermore, in silico tools (SIFT and Mutation taster) used for variant predictions, revealed this mutation to be strongly deleterious and induces nonsense-mediated mRNA decay (NMD) which degrades the mRNA due to premature termination codon [11]. This novel pathogenic LDLR mutation was then screened in the family using PCR amplification and Sanger sequencing. Blood samples were available for six additional family members (I.2, II.5, II.6, II.7, II.8, III.4) including the proband and interestingly, except the clinically unaffected member II.6, rest all clinically affected five members (including proband) harbored the same c.743_744delinsAA heterozygous LDLR mutation.