Mutations in our genes responsible for higher iron loads

Diagnosing a genetic predisposition for a higher iron load could prevent damage to tissues and organs in the future, which is a complication of long-term higher iron loads. My earlier article summarizes higher iron load with breast cancer and more.

Patent Several scientists have discovered mutations in a gene recently named SLC40A1. One recently awarded United States patent to an Italian scientist (patent number 7718785) awarded May 2010, describes higher iron load issues in general, the existing literature published in the field and the diagnostic tools available in the future should companies be interested in marketing them. Hopefully, this patient pool is taken care of by discerning family physicians.

Detailed description of Invention: The authors of the present invention have identified new mutations in the SLC40A1 gene (Solute Carrier Family) coding for ferroportin 1 (IREG1 or MTP1), previously also named SLC11A3, genetically linked to Hereditary Hemochromatosis or to an Impaired non-HFE iron homeostasis (Hereditary Hemochromatosis).

Mutations cause aminoacid substitutions in the corresponding protein whose expression as a mutated form causes abnormal iron overload in carrier subjects. From the functional point of view indeed ferroportin has a key role in at least two different but correlated aspects of iron homeostasis: in the enterocytes ferroportin causes the uptake of iron introduced by diet, whereas in the reticular endothelial cells particularly in macrophages, it causes the iron release from intracellular stores. Said new mutations are responsible for the Hemochromatosis and are characterized by clinical traits at least partially similar to those already described in Pietrangelo et al. New England Journal of Medicine 1999, 341 (10): 725-732, caused by the mutation of the aminoacid 77 in the ferroportin sequence (A77D mutation) described In WO 02/033119.

Therefore, a first aspect of the invention refers to polymorphic polynucleotides related to mutated SLC40A1 sequences, which encode for mutated forms of the wild type ferroportin 1 and in particular to at least one of the following polymorphisms: polymorphism of the nucleotide corresponding to the nucleotide 238 of the IDN 1 sequence, preferably related to the substitution of a Guanine with an Adenosine (G.fwdarw.A), which causes the replacement of aminoacid 80 with an aminoacid different from Glycine and preferably with Serine (G80S) in the coded protein: the cDNA derived from such polymorphic gene has preferably the IDN3 sequence; polymorphism of the nucleotide corresponding to the nucleotide 521 of the IDN 1 sequence, preferably related to the substitution of an Adenine with a Tymine (A.fwdarw.T), which causes the replacement of aminoacid 174 with an aminoacid different from Asparagine and preferably with Isoleucine (N174I) in the coded protein: the cDNA derived from such polymorphic gene has preferably the IDN5 sequence; polymorphism of the nucleotide corresponding to the nucleotide 744 of the IDN1 sequence, preferably related to the substitution of a Guanine with a Tymine (G.fwdarw.T), which causes the replacement of amino acid 248 with an amino acid different from Glutamine and preferably with Hystidine (Q248H) in the coded protein: the cDNA derived from such polymorphic gene has preferably the IDN7 sequence; or their oligonucleotide fragments comprising the polymorphic nucleotide of at least 10 base pairs.

Example 1

Identification of the Mutations in the Ferroportin Gene

Genomic DNA of index cases, of family members and of control subjects, was extracted from leucocytes obtained by blood samples using a blood DNA extraction kit (Quiagen).

Obtained DNA was then amplified by PCR using primers pair able to amply the whole coding region including exon/intron boundary regions of the ferroportin.

Primers pairs used herein are the following:

TABLE-US-00001 Exon 1: Fw.1: 5′-GGTGCTATCTCCAGTTCCTT-3′ (IDN 9) Rv.1: 5′-GTTCACAGCAGAGCCACATT-3′ (IDN 10) Exon 2: Fw.2: 5′-CAGCTCATTAAGTGACTACCATCGC-3′ (IDN 11) Rv.2: 5′-GGCTTAATACAACTGGCTAGAACG-3′ (IDN 12) Exon 3: Fw.3: 5′-CATAATGTAGCCAGGAAGTGCCC-3′ (IDN 13) Rv.3: 5′-TCCAGAGGTGGTGCCATCTAAG-3′ (IDN 14) Exon 4: Fw.4: 5′-GAGACATTTTGATGTAATGTACAC-3′ (IDN 15) Rv.4: 5′-CTACCAGATATTCAATTTTCTGCC-3′ (IDN 16) Exon 5: Fw.5: 5′-CCACCAAAGACTATTTTAAACTGC-3′ (IDN 17) Rv.5: 5′-TCACCACCGATTTAAAGTGAATCC-3′ (IDN 18) Exon 6: Fw.6: 5′-GTATTGTGTAAATGGGCAGTCTC-3′ (IDN 19) Rv.6: 5′-CCCCACTGGTAATAATAAAACCTG-3′ (IDN 20) Exon 7: Fw.7: 5′-GGCTTTTATTTCTACATGTCCTCC-3′ (IDN 21) Rv.7: 5′-ACATTTAGGGAACATTTCAGATC-3′ (IDN 22) Exon 8: Fw.8: 5′-AAGGTGACTTAAAGACAGTCAGGC-3′ (IDN 23) Rv.8: 5′-GCTGACTTAGGTTTCCTAAACAGC-3′ (IDN 24)

The amplification of the regions corresponding to each exon was performed as follows: 200 ng of genomic DNA were amplified in 50 .mu.l of reaction buffer 1.times. containing dNTPs 200 .mu.M, MgCl2 1.5 mM, 25 pmoles of each of the aforementioned oligonucleotides, 1 U of Taq polymerase (Applied Biosystems).

In the amplification reaction was used a program of 30 cycles, each characterized by the following thermal profile:

94.degree. C. for 1 minute,

58.degree. C. for 40 seconds,

75.degree. C. for 5 minutes.

Obtained fragments were purified and sequenced by automatic sequencing with the Backman Coulter Sequencer. The sequence analysis allowed the identification of the G80S mutation in the exon 3 and the N174I and Q248H mutations in the exon 6, as compared to the wild type sequence (GenBank accession number; AF231121) that was not detected in any of the control subjects.

A further evaluation of the mutations was performed by the digestion of an aliquote of the same first PCR product with endonucleases whose restriction site is modified by the nucleotide substitution.

In particular, the Q248H mutation was verified by digestion according to the Manufacturers Instructions (New England Biolabs), the first product of PCR with the PvuII enzyme, which cut into GC in the 5’CAGCTG 3′ sequence. The G.fwdarw.T base substitution in the mutated sequence removes the restriction site of the enzyme.

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