Tomoko Maeda

Chronological difference in walking impairment among Japanese group A xeroderma pigmentosum (XP‐A) patients with various combinations of mutation sites

Almost all Japanese group A xeroderma pigmentosum (XP‐A) patients have nonsense and/or nonsense codon‐leading mutations in the XP group A (XPA) gene, and develop neurological abnormalities. Walking ability is one of the most important neuromuscular functions of the patients, because it determines their daily activities. We studied the correlation between the various combinations of mutations found by PCR‐RFLP in Japanese XP‐A patients and their chronological walking impairment. We classified these patients into six groups. Group I: A patient who was homozygous for the mutation at codon 116 in exon 3 (Type 1 mutation) could never walk unaided. Group III: Typical patients who were homozygous for the mutation at intron 3 (Type 2 mutation) could walk unaided till 7–16 years of age. Group V: Patients who were compound heterozygous for Type 2 mutation and for the mutation at codon 228 in exon 6 (Type 3 mutation) began to develop some walking difficulty at 5–13 years of age and became unable to walk at 25–28 years of age. Group VI: A patient who was homozygous for Type 3 mutation could walk unaided without any difficulty till the age of 21. The walking ability of group II and IV patients is not known yet.

Severe neurological abnormalities associated with a mutation in the zinc-finger domain in a group A xeroderma pigmentosum patient

All the reported Japanese patients with group A xeroderma pigmentosum (XP) have two or three mutations at codon 116 in exon 3, codon 228 in exon 6, and the splicing acceptor site of intron 3 of XP group A complementing (XPAC) gene. A homozygote (XP390S) with a nonsense mutation at codon 228 has less severe neurological abnormalities than patients with the splicing mutation at the acceptor site of intron 3. As homozygotes for the nonsense mutation at codon 116, which truncates a carboxyl‐terminal site of XPAC protein at an early part of its zinc‐finger domain, have not been reported previously, the possible severity of associated neurological abnormalities was not known. We report a group A XP patient, XP180S, who had neurological abnormalities which were more severe than those in patients homozygous for the splicing mutation. The polymerase chain reaction product from exon 3 of the patients XPAC gene was digested completely into three fragments by MseI restriction endonuclease. Thus, the patient was homozygous for the mutation at codon 116.

Compound heterozygous group A xeroderma pigmentosum patient with a novel mutation and an inherited reciprocal translocation

The severity of neurological abnormalities in Japanese group A xeroderma pigmentosum (XP‐A) patients correlates with the sites of non‐sense mutation in the XP‐A gene. We describe a patient who presented with a more severe photosensitivity and neurological abnormality than those in typical Japanese XP‐A patients with a splicing mutation in intron 3. The patient was compound heterozygous for the splicing mutation in intron 3, which resulted in formation of a non‐sense codon in exon 4, and a novel non‐sense mutation at codon 208 in exon 5, a C to T transition creating a stop codon TAG. Although the combination of these mutations might have been thought to cause only mild neurological signs, the longer truncated XP‐A proteins than those of typical XP‐A patients may have resulted in severe neurological symptoms. This phenomenon may be explained by a translocation of chromosome (1;10)(q25·3;q22·3) inherited from his father.

An Improved Protocol for mRNA Quantification After Fluorescence-Activated Cell Sorting with an Increased Signal to Noise Ratio in Flow Cytometry

We established a method to analyze cells collected by fluorescence-activated cell sorting (FACS) named mRNA quantification after FACS (FACS-mQ), in which cells are labeled with a fluorescent dye in a manner that minimizes RNA degradation, and then cells sorted by FACS are examined by analyzing their gene expression profile. In this study, we established a modified protocol to analyze molecules with a low expression level, such as N-cadherin and thyroid transcription factor, by improving the signal to noise ratio in flow cytometry. Use of a fluorophore-conjugated second antibody and the appropriate choice of a fluorescence dye showed a marked increase in the signal to noise ratio. Use of the Can Get Signal Immunostain in diluting antibodies shortened the reaction time. In real-time reverse transcription-PCR, a significant decrease in the copy number of intracellular mRNAs was not observed after in-tube immunostaining. These results indicated that the present protocol is useful for separating and analyzing cells by FACS-mQ, targeting a molecule with a low expression level.