This invention relates to a set of novel primers used for detection of the 8bp deletion mutation in exon 3 of the 21 Hydroxylase (CYP 21) gene. The mutation is one of the common causes of classical salt wasting Congenital Adrenal Hyperplasia (CAH), a potentially lethal condition.

This invention further relates to a PCR based detection technique for the amplification of the 8bp region and control region in exon 3 of the 21 Hydroxylase (CYP 21) gene.

BACKGROUND OF THE INVENTION:

CAH is a treatable genetic condition prevalent worldwide. It is an autosomal recessive disorder with prevalence varying globally between 1 : 10000 to 1 : 15000 live births. No estimates are however available in the Indian context. Potentially CAH is a fatal disorder and early detection ensures early treatment, helping the affected individual lead a normal life.

The condition arises mainly due to mutations in the 21 -Hydroxylase gene (CYP 21) and detection of these mutations has provided accurate basis for its early diagnosis. A number of DNA based diagnostic techniques have been employed over the years by various workers with varying degrees of sensitivity viz. Southern blotting, PCR-RFLP, Real Time PCR, ACRS, MLPA, DGGE and reverse-hybridization test strip-based assay (CAH Strip Assay). Other techniques such as multiplex minisequencing, multiplexed peptide mass signature genotyping (PMSG) and LC-MS/MS have also been used by investigators with varying degrees of specificity. Emergence of the DNA sequencing techniques for mutation analysis in recent years has now provided the gold standard for this diagnosis. US patent number US8153962 and US8415616 discloses to a Mass spectrometry based method for detecting amounts of one or more CAH panel analyses (i.e., pregnenolone, 17-OH pregnenolone, progesterone, 17-OH progesterone, dehydroepiandrosterone (DHEA), androstenedione, testosterone, deoxycortocisterone, 1 1-deoxycortisol and Cortisol).

The method is not suitable for general application as it requires specially trained skills in handling the Mass Spectrometer and its data interpretation. The hormone assays, except 17- OH progesterone are not found suitable for CAH diagnosis as the normal ranges have not been established for different populations at various age groups. Additionally, the equipment as well as chemicals have to be sourced from other countries, adding to its costs.

US patent number US4230684 discloses the patent pertains to a filter paper method that can be used for quantitating steroids such as 17- Hydroxyprogesterone for diagnosis of CAH. The steroid is eluted from the filter paper and quatitated by Radiommunoassay. The method is particularly suitable for screening of newborns.

Filter paper techniques along with RIA as described here have been sued in new born screening. However, it requires the use of hazardous radioactive material. The borderline results need to be reanalysed and confirmed through DNA sequencing.

US patent number US5807678 discloses to the specific diagnosis of Congenital Lipoid Adrenal Hyperplasia through mutation analysis of the gene for steroid acute regulatory protein (StAR) by RFLP, nucleic acid hyrbridisation or nucleotide sequencing. However, these techniques, although sensitive, are highly cost as well as labour intensive, demanding an advanced laboratory set up with state of the art equipment facility.

Accordingly there is long felt need to establish an improved method for detection of the 8bp deletion mutation in exon 3 of CYP 21 gene which is cost effective and labour intensive and applicable to low resource settings as well.

The present invention meets the long felt need by developing a novel primers and a novel PCR based method for detecting the 8bp deletion mutation in gene which is simple, quick and widely acceptable in smaller laboratories which do not have the high end equipment support.

OBJECTS OF THE INVENTION:

It is therefore an object of this invention to propose a set of novel primers and PCR based technique and primers for the detection of 8bp deletion mutation in CAH, which is simple and less labour intensive.

It is a further object of this invention to propose a set of novel primers and PCR based technique and primers for the detection of 8bp deletion mutation in CAH, which is cost-effective and can be conducted in set-ups lacking high end equipment support.

These and other objects and advantages of the invention will be apparent from the ensuing description, when read in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

Fig. 1: Standardization of Biplex PCR for Detection of 8bp Deletion: Lane 1: lOObp DNA molecular weight ladder. Lanes 2,3,4,5: Amplification at annealing temperatures of 45°C, 47°C, 48°C degrees and 50°C respectively in a normal sample DNA. Optimum amplification was obtained using annealing at 48°C. Amplicon at 340 bp corresponds to the 8bp locus and the amplicon at 520 bp corresponds to the internal control region.

Fig. 2: Detection of 8bp deletion. The Biplex PCR test in a sample DNA with 8bp deletion: No amplification is seen corresponding to the 8bp locus and amplification of only the internal control region of 520 bp (Lanes 2 and 3) is seen.

Fig. 3: DNA Sequence of 8bp locus in the normal control sample.

Fig. 4: DNA sequence of the sample with 8bp deletion. Deletion locus marked by arrow.

Fig. 5: Fragment analysis of a normal control sample for the 8bp deletion locus. Presence of the two peaks at 150bp and 158bp denotes existence of the 8bp region in active gene.

Fig. 6: Fragment analysis of the test sample for the 8bp deletion locus. Absence of the peak at 158 bp denotes deletion of the 8bp region in active gene. DETAILED DESCRIPTION OF THE INVENTION:

According to this invention is provided a novel set of primets and also a PCR based technique for detection of the 8bp deletion mutation in exon 3 of the 21 Hydroxylase (CYP 21) gene. The mutation is one of the common causes of classical salt wasting Congenital Adrenal Hyperplasia (CAH), a potentially lethal condition and primers pairs therefore.

The Biplex PCR technique has been developed with an aim to provide a indigenous diagnostic method applicable in low resource setting.

In accordance with this invention, Biplex PCR technique has been developed by us to specifically detect one of the commonly found causative mutations in CAH viz. 8bp deletion in 21 -Hydroxylase gene. This 8bp deletion is null mutation causing 100% inactivation of the 21- Hydroxylase activity, causing the severe salt wasting form of CAH. The mutation has been reported worldwide in different ethnic groups with reported frequencies ranging from 4% to 26%. The techniques used were Southern blotting, RFLP, nested PCR and fragment analysis which have varying degrees of sensitivity. With similar objective, we carried out an analysis in our Laboratory in a group of 70 clinically defined Indian cases of CAH using DNA sequencing analysis. Our results showed that the 8bp deletion mutation was responsible for about 20% of the clinically suspected cases of CAH (Unpublished observation). However, this DNA sequencing approach, being cost and labour intensive, cannot be applied in low resource settings. A PCR based diagnostic technique with an inbuilt confirmatory step would find wide acceptance in smaller laboratories which do not have the high end equipment support. The PCR based confirmatory test has been developed by us which can precisely detect the 8bp deletion mutation. The test to perform and enables accurate detection, comparable to DNA sequencing.

The test uses a Biplex approach involving two primer sets (Table 1).

Sequences of these primers are designed so as to provide two distinct bands of 340bp for the region encompassing the 8bp locus of the active gene and 520bp representing internal control region. The internal control region has been selected to represent a non-variable segment which includes Exon 9 and part of Exon 10 of the active CYP21 gene. Selection of this non-variable region is based on our mutation analysis by sequencing carried out earlier, in about 70 clinically diagnosed Indian cases of CAH. Temperature profile for the PCR reaction has been optimised using a normal sample, such that it yields the two specific bands of 340bp and 520bp as seen in Fig. 1. A reaction volume of 50 μΐ was used containing 200 ng of genomic DNA, 1.5 mM MgCb, 0.2 mM dNTPs, 200 mM (NH ₄ ) ₂ S0 ₄ , 750 mM Tris-HCl (pH 8.8), 0.1% Tween, 2 units of Taq polymerase (Fermentas, Life Sciences) and 50 pm of primers. The final temperature profile used was as follows: 96°C for 5 min for initial denaturation followed by 30 cycles of 95°C for 1 min, 48°C for 1 min, and 72°C for 1 min and final extension at 72°C for 10 min.. Fig.2 shows the PCR analysis in a known case of CAH due to deletion of the 8bp region. PCR analysis in this sample showed a single band of 520bp corresponding to the control fragment. This amplification of the control fragment alone without that corresponding to the 8bp locus indicates homozygous deletion of the 8bp site. DNA sequencing was used to reconfirm the 8bp locus in the normal sample as well as the 8bp deletion in the CAH sample (Fig 3, 4). A secondary confirmation of the results was also carried out by the method of fragment analysis. For fragment analysis, primers were designed such that in presence of 8bp region within the active gene, a peak of 158bp is obtained, along with a second peak of 150bp corresponding to the same locus in the pseudo gene. Fig.5 shows fragment analysis of the normal sample with peaks of both 150bp for pseudo gene as well as 158bp for active gene. Similar analysis in the CAH sample showed only a single peak of 150bp corresponding to the pseudo gene (Fig.6). Absence of the 158 bp peak in this case confirms homozygous deletion of the 8bp region as observed through PCR as well as DNA sequencing. Thus the existence or deletion of the 8bp region as determined through the novel PCR technique was validated both by DNA sequencing as well as by fragment analysis.