GENETIC INVESTIGATION OF COMPLEX INHERITANCE 
Jean-Marc Lalouel, M.D., D.Sc, Investigator 
Research in Dr. Lalouel's laboratory focuses on 
two topics: 1) the identification of genetic factors 
that account for familial aggregation of disorders of 
lipid metabolism and early occurrences of myocar- 
dial infarction and 2) the construction of genetic 
maps of human chromosomes, a project pursued in 
collaboration with Dr. Raymond L. White (HHMI, 
University of Utah). The major focus of research 
during the past year has been an investigation of 
lipoprotein lipase (LPL) deficiency in relation to 
hypertriglyceridemia. 
Complex lipoprotein phenotypes are commonly 
observed among relatives of patients with early cor- 
onary disease, with various patterns of hypercho- 
lesterolemia and/or hypertriglyceridemia. Complex 
lipoprotein profiles have been considered to result 
either from the variable expression of a single-gene 
defect or from the independent contribution of two 
or more genes, but etiological heterogeneity further 
confuses the issue. The investigation of a large kin- 
dred expressing complex lipoprotein phenotypes, 
K659, yielded preliminary results in support of 
the second hypothesis: one gene accounted for hy- 
percholesterolemia, while hypertriglyceridemia de- 
pended on other factors. Because chylomicronemia 
was noted in one pedigree member, LPL, which is a 
key enzyme in the delivery of fatty acids to periph- 
eral tissues, stood as a possible candidate for the 
cause of the defect. Deficient LPL activity is found in a 
rare form of familial hyperchylomicronemia that is 
inherited as an autosomal recessive trait. However, 
the hetero2ygous state remains poorly character- 
ized. Are heterozygotes particularly prone to hyper- 
triglyceridemia? Can heterozygosity for a defect at 
the LPL locus account for some of the commonly 
observed forms of familial hypertriglyceridemia? 
Another pedigree, K2003, ascertained through a 
patient with documented deficiency of LPL activity, 
was investigated in an effort to address the above 
questions. In collaboration with Dr. Lalouel's 
group, Dr. P. H. Iverius at the Veterans Administra- 
tion Hospital in Salt Lake City confirmed by immu- 
noassay that LPL was present in this proband. The 
sequence of human LPL cDNA had just been re- 
ported in the literature, but no mutation had been 
identified. The mRNA prepared from adipose tissue 
of this patient was used to synthesize cDNA, from 
which the complete coding region of LPL was am- 
plified and cloned. Eight independent clones were 
sequenced. A single-nucleotide difference was iden- 
tified in the patient's LPL cDNA, leading to the 
substitution of a glutamic acid for a glycine. The pa- 
tient was found to be homozygous for this mu- 
tation by dot-blot hybridization with specific oligo- 
nucleotides, and both parents were confirmed 
heterozygotes. To establish the functional signifi- 
cance of this molecular variant, the mutation was 
reproduced by oligonucleotide-directed in vitro 
mutagenesis, and transient expression of both nor- 
mal and mutant genes was achieved by transfection 
of COS cells after cloning into the expression vec- 
tor pSVL. Normal activity was recovered both in 
medium and in cell extracts after transfection with 
the normal sequence; no LPL activity could be dem- 
onstrated after transfection with the mutant se- 
quence, although concentrations of immunoreac- 
tive material were similar to those obtained with 
the normal sequence. These results confirmed that 
the mutation identified in the patient led to the 
production of an inactive product. 
The identification of this mutation permitted de- 
termination of the genotype of relatives of the pro- 
band by hybridization of their individual genomic 
DNAs, amplified by the polymerase chain reaction, 
to specific oligonucleotides; it also permitted test- 
ing for a relationship between carrier status for the 
mutation and the presence of hypertriglyceridemia 
noted in multiple members of the pedigree. An 
association was found in an initial screen of 27 rela- 
tives of the proband; this association was confirmed 
by extending the original pedigree to include 61 
relatives. However, only 50-60% of the carriers 
of this mutation expressed hypertriglyceridemia. 
Therefore, although individuals heterozygous for 
an allele encoding a functionally deficient LPL en- 
zyme were prone to exhibit hypertriglyceridemia, it 
appeared that other factors were necessary to in- 
duce the expression of this phenotype. These find- 
ings may be relevant to hypotheses being advanced 
for the inheritance of familial hypertriglyceridemia 
and related disorders. 
The hypothesis was subsequently entertained 
that the chylomicronemia observed in one member 
of pedigree K659 may result from LPL deficiency. 
Dr. Iverius established that LPL activity in the 
plasma of this subject after heparin injection was 
below the fifth percentile of normal controls. The 
strategy outlined above for K2003, applied to clone 
and sequence the entire coding region of the LPL 
gene in this subject, led to the identification of an- 
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