PUMA, a pro-apoptotic gene, as a novel molecular biomarker for TNFα-induced human islet damage

Patent metadata

Field	Value
Publication number	US-9758826-B2
Application number	US-201113163326-A
Country	US
Kind code	B2
Filing date	Jun 17, 2011
Priority date	Jun 24, 2010
Publication date	Sep 12, 2017
Grant date	Sep 12, 2017

Abstract

p53-upregulated modulator of apoptosis (PUMA) is a biomarker associated with islet cell health. If PUMA is low, islet cells are typically healthy. If PUMA is high, islet cells are typically unhealthy or dying. PUMA may be measured by either measuring its nucleic or amino acid. PUMA mRNA may be induced by TNF-α stimulation in a time- and dose-dependent manner and β cell apoptosis is induced through a mitochondrial pathway. TNF-α significantly inhibited glucose-induced preproinsulin precursor mRNA synthesis. Such β cell stress signaling in human islets indicates overall state of islet health and, ultimately, the risk of onset and/or degree of severity of both type 1 and type 2 diabetes mellitus.

First claim

The invention claimed is:

1. A method of treating hyperglycemia or diabetes in a subject comprising:
determining whether an islet is a candidate for transplant, comprising:
quantifying an expression level of p53 upregulated modulator of apoptosis (PUMA) messenger RNA (mRNA) comprising:
reverse transcribing an RNA sample from the islet, forming a first cDNA sample, and
amplifying the first cDNA sample to determine the expression level of PUMA mRNA, wherein the expression level of PUMA is determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR);

quantifying an expression level of pre-spliced preproinsulin mRNA comprising;
reverse transcribing the RNA sample from the islet, forming a second cDNA sample, and
amplifying the second cDNA sample to determine the expression level of pre-spliced preproinsulin mRNA, wherein the expression level of pre-spliced preproinsulin mRNA is determined by qRT-PCR;

determining the islet is a candidate for transplant when the expression level of pre-spliced preproinsulin mRNA is greater than the expression level of PUMA mRNA; and

transplanting islets that are equivalent to the islet determined to be a candidate for transplant into the subject to treat the hyperglycemia or diabetes.

2. The method of claim 1 , wherein the expression level of PUMA is normalized to an expression level of a control gene and the expression level of pre-spliced preproinsulin mRNA is normalized to an expression level of pre- and post-spliced preproinsulin mRNA.

3. The method of claim 1 , wherein the islet is a candidate for transplant when the expression level of PUMA mRNA is less than a cutoff value of 0.5.

4. The method of claim 1 , wherein the subject is suffering from hyperglycemia or diabetes and the transplanted islets reverse the hyperglycemia or diabetes.

5. The method of claim 1 , wherein one or more nucleotide probe pairs are used to perform qRT-PCR of pre-spliced preproinsulin mRNA.

6. The method of claim 5 , wherein the one or more nucleotide probe pairs comprise:
AGGTGGGCTCAGGATTCCA (SEQ ID NO.1) (In1 upstream) and TCACCCCCACATGCTTCAC (SEQ ID NO.2) (In1 downstream);
ACTCGCCCCTCAAACAAATG (SEQ ID NO.3) (In2 upstream) and TGAATCTGCGGTCATCAAATG (SEQ ID NO.4) (In2 downstream);
CTCTGCCTCGCCGCTGTTC (SEQ ID NO.5) (In2Ex3 upstream) and TCCACAATGCCACGCTTCTG (SEQ ID NO.6) (In2Ex3 downstream);
GCAGCCTTTGTGAACCAACA (SEQ ID NO.7) (Ex2a upstream) and TTCCCCGCACACTAGGTAGAGA (SEQ ID NO.8) (Ex2a downstream);
GGGAACGAGGCTTCTTCTACAC (SEQ ID NO.9) (Ex2b upstream) and CCACAATGCCACGCTTCTG (SEQ ID NO.10) (Ex2b downstream); and
CATTGTGGAACAATGCTGTACCA (SEQ ID NO.11) (Ex3 upstream) and GCCTGCGGGCTGCGTCTA (SEQ ID NO.12) (Ex3 downstream).

Assignees

• Mullen Yoko
• Mitsuhashi Masato
• Omori Keiko
• Hope City
• Hitachi Chemical Res Center Inc
• Hitachi Chemical Co Ltd

Inventors

• Mullen Yoko
• Mitsuhashi Masato
• Omori Keiko

Classifications

• G01N2333/4748

  p53 · CPC title

• G01N33/507

  Pancreatic cells · CPC title

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/136

  Screening for pharmacological compounds · CPC title