1. XERODERMA PIGMENTOSUM We have been examining XP patients at NIH since 1971. We performed long term follow-up studies of cancer, of hearing loss and of eye abnormalities in XP patients. We found that the XP patients under age 20 years had a 10,000-fold increased risk of non-melanoma skin cancer (NMSK) and a 2000-fold increased risk of melanoma. In our natural history protocol we ascertained and intensively examined 105 XP or Cockayne Syndrome (CS) patients and 206 family members at NIH and are collaborating with international researchers to study additional patients. We published detailed autopsy reports of 4 XP patients - including the first autopsy of an XP-D patient. The XP-A and XP-D patients were adults with severe neurological degeneration and were found to have infant sized brains. We have now identified mutations in all of the 9 currently known DNA nucleotide excision repair (NER) genes (XPA, XPB, XPC, XPD, XPE, XPF, XPG, ERCC1 and TTDA), in the error-prone polymerase, pol eta and in TTDN1 in nearly 200 XP or TTD patients. We have established several hundred carefully documented cell lines and contributed them to cell banks for use of the general scientific community. We are using array comparative genome hybridization (aCGH) and whole exome sequencing to look for mutations in other genes in cells from XP and TTD patients who do not have mutations in these genes. Our laboratory is the major center in the US for basic, clinical and translational expertise concerning DNA repair related disorders. We are actively seeking and have developed the expertise to recognize unusual patients who have unique disease features that provide insights into the functioning of DNA repair genes. These studies have enabled us to identify some remarkable XP patients and better characterize different mechanisms of DNA repair. For example, we found XPC patients with splice lariat branch point mutations who had 3 to 5% of XPC mRNA but mild disease indicating that only a small amount of XPC is sufficient for some cancer protection. We found that NER proteins accumulate and persist at sites of DNA damage in XP-B cells. In contrast, these NER proteins rapidly accumulate but fail to persist in UV damaged XP-E cells. We found that the melanomas and nevi in the XP patients were different from those in the general population both clinically and histologically. They had a high proportion of mutations in the PTEN tumor suppressor gene (90% UV type), a lower frequency of mutations in BRAF, NRAS or KIT and rarely had the BRAF V600E mutation found in the general population. We plan to develop a protocol to attempt to reduce the melanoma risk in the XP patients by inhibition of the PTEN, mTOR pathway. About 12% of genetic diseases involve premature stop codons (PTC). We developed sensitive assay systems to detect readthrough of premature stop codons using cells from our collection with PTC in the XPC DNA repair gene. We found the aminoglycoside, gentamycin, can improve DNA repair in selected patients, permitting precise targeting of therapy to responsive individual cell lines. We are performing pre-clinical tests to determine if topical aminoglycosides can increase DNA repair without systemic toxicity. We are collaborating with NCI epidemiologists to study clinically normal family members of XP patients to determine if XP heterozygotes, who are much more frequent than XP patients, have increased cancer risk. We have 257 XP patients and family members in 54 families enrolled in this protocol. Our investigation of Big Data in the gnomAD database of more than 200,000 alleles revealed an unexpectedly high frequency of 2 XP causing missense variants, suggesting that there may be thousands of patients who are homozygous for these XP causing mutations in the US. Unsuspected mutations in known genes with a predisposition for skin cancer may be responsible for some of the high frequency of skin cancers in the general population. It is possible that variants in other genes modify the phenotype so that the usual features of XP are not apparent.. 2. TRICHOTHIODYSTROPHY In contrast to the profound environmental influence of sun exposure on XP, TTD is a disease of altered development. In our current natural history protocol we have ascertained and intensively examined 58 TTD or XP/TTD patients and 116 TTD family members. This is the largest cohort of patients with this rare disorder in the world.We found that TTD patients with mutations in the TTDN1 gene had a distinct phenotype. In collaboration with gynecologists and epidemiologists we published 3 studies on pregnancy abnormalities in TTD. There was a high frequency of gestational abnormalities in pregnancies resulting in TTD affected offspring compared to pregnancies resulting in unaffected offspring from the same mothers or to the general population. These pregnancy complications were present only in pregnancies that had XP-D mutations that resulted in TTD offspring but not in XP-D mutations resulting in XP offspring. These observations provide important insights into the role of DNA repair genes in human pregnancy and fetal development. In collaboration with the ophthalmologists, we published a detailed report of the eye findings of 32 TTD patients we studied from 2001 to 2010. Infantile cataracts were present in more than half of these patients. We documented the severe growth retardation in children with TTD. Several young children with TTD have lost the ability to walk due to aseptic necrosis of the femoral head. Unfortunately, surgical treatment was followed by a series of post-operative complications leading to death. We are collaborating with radiologists to attempt to determine early signs of this problem and guide management. We have also found patients with features of more than one disease. Thus we identified 12 XP/TTD patients who have features of both XP and TTD and increased cancer risk. We are attempting to further define this entity by use of laboratory testing of DNA repair. In collaboration with colleagues in Italy, we identified mutations in a transcription associated gene (GTF2E2) as causing TTD in two patients. This closely links TTD to transcription defects rather than DNA repair defects.
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