Analysis of DNA ligase IV mutations found in LIG4 syndrome patients: the impact of two linked polymorphisms

Abstract

LIG4 syndrome patients have hypomorphic mutations in DNA ligase IV. Although four of the five identified patients display immunodeficiency and developmental delay, one patient was developmentally normal. The developmentally normal patient had the same homozygous mutation (R278H) in DNA ligase IV as one of the more severely affected patients, who additionally had two linked polymorphisms. Here, we examine the impact of the mutations and polymorphisms identified in the LIG4 syndrome patients. Examination of recombinant mutant proteins shows that the severity of the clinical features correlates with the level of residual ligase activity. The polymorphisms decrease the activity of DNA ligase IV by approximately 2-fold. When combined with the otherwise mild R278H mutation, the activity is reduced to a level similar to other LIG4 patients who display immunodeficiency and developmental delay. This demonstrates how coupling of a mutation and polymorphism can have a marked impact on protein function and provides an example where a polymorphism may have influenced clinical outcome. Analysis of additional mutational changes in LIG4 syndrome (R580X, R814X and G469E) have led to the identification of a nuclear localization signal in DNA ligase IV and sites impacting upon DNA ligase IV adenylation.

Pyrimidine pool imbalance induced by BLM helicase deficiency contributes to genetic instability in Bloom syndrome

“…BS-V and BS-CDA cell lines were obtained by transfecting BS cells with an empty pCI-puro vector 25 , or with the same vector containing the full-length CDA cDNA (NM001785), using JetPEI reagent. After 48 h, selection was carried out with 0.2 µg ml − 1 puromycin (Invivogen) and 500 µg ml − 1 G418 (Invitrogen).…”

Severe combined immunodeficiency and microcephaly in siblings with hypomorphic mutations in DNA ligase IV

“…Both patients had a distinct phenotype, since one developed leukemia [25], while the second patient exhibited a developmental delay and mild immunodeficiency [20]. The latter patient carried additional homozygous mutations (A3V and T9I), previously described as polymorphisms, which have been found to dramatically reduce the 10% residual in vitro ligase activity of the R278H Lig4 mutant using a RH278H/A3V/T9I Lig4 expression construct [28]. The impact of these two linked mutations may explain the different patients' phenotypes.…”

DNA double-strand break repair and development

“…Additional LIG4 syndrome individuals have since been identified and display varying penetrance of the phenotype, due to differing hypomorphic mutations. For instance, an individual carrying the R278H mutation in addition to the N-terminal polymorphisms A3V and T9I presented with microcephaly, pancytopenia and developmental delay (O'Driscoll et al, 2001;Girard et al, 2004). In vitro studies revealed no residual LIG4 adenylation or ligation activity in that combination of mutations (Girard et al, 2004).…”

“…For instance, an individual carrying the R278H mutation in addition to the N-terminal polymorphisms A3V and T9I presented with microcephaly, pancytopenia and developmental delay (O'Driscoll et al, 2001;Girard et al, 2004). In vitro studies revealed no residual LIG4 adenylation or ligation activity in that combination of mutations (Girard et al, 2004). Two siblings who presented with SCID, growth defects and microcephaly were found to harbor compound heterozygous mutations conferring a Q280R substitution on one allele and a K424 frame-shift mutation that resulted in truncation of the C terminus on the other allele (Buck et al, 2006b).…”