RU2315310C1 - Method for diagnosing 3-m syndrome yakut population - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves carrying out polymerization chain reaction study with original primers for detecting 4582 insT mutation (Arg1528Ser) in cullin7 gene ((CUL7), characteristic of Yakut population with Restriction Fragment Length Polymorphism-analysis being next done and restrictase HinfI being used. 218 n.p. and 175, 26 n.p. long fragments availability is thought to be pointer to mutant gene carriage in homozygotic state (disease). 218, 175, 114, 104 and 26 n.p. long fragments availability is thought to be pointer to mutant gene carriage in heterozygotic state (healthy mutation carrier). 175, 114, 104 and 26 n.p. long fragments availability is considered to be pointer to norm (healthy).

EFFECT: rapid and accurate diagnosis method.

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Description

The scope refers to medicine, namely to molecular genetic methods for the diagnosis of hereditary diseases.

The closest analogue of the diagnostic method for 3-M syndrome in the Yakut population is the molecular genetic method for identifying a mutation in the Cullin 7 gene (CUL7) in the 3-M syndrome, including PCR determination in the CUL7 gene, mapping in chromosome 6p21.1., 25 different mutations in subjects from 29 families with 3-M syndrome. As a result, nonsense and missense mutations R1445X and H1464P were found, which play an important role in the inhibition of intrauterine growth in humans (Huber C. et al. Identification of mutations in 3-M syndrome / Nat. Genet. - 2005. - Oct.37 (10 ). - P.1119-24).

The diagnostic method for 3-M syndrome described by Huber et al. Was used to diagnose 3-M syndrome in the Yakut population in 39 patients with 3-M syndrome. None of the mutations described in this source is characteristic of the Yakut population. Another mutation 4582 insT (Arg 1528 Ser), characteristic of the Yakut population, was revealed in the CUL7 gene. This method does not allow to identify the mutation 4582 insT (Arg 1528 Ser), characteristic of the Yakut population, thereby does not allow to make an accurate and final diagnosis of the disease, to give an accurate genetic risk of the disease for offspring in the family.

In order to eliminate the shortcomings of the method described by Huber et al., A method for the diagnosis of 3-M syndrome by molecular genetic analysis by PCR reaction using original F-GTATGCACTTAGGGCCAGAG primers is proposed; R-CACACTCCTACAGGCTACAC for detecting the 4582 insT mutation (Arg 1528Ser) in the Cullin 7 gene (CUL7) characteristic of the Yakut population, followed by analysis of restriction fragment length polymorphism using HinfI endonuclease and in the presence of 218 bp fragments and 175, 26 bp diagnose carriage of a mutant gene in a homozygous state (patient), in the presence of fragments - 218, 175, 114, 104 and 26 bp Carriage of a mutant gene in a heterozygous state (a healthy carrier of a mutation) is diagnosed, and with fragments length 175, 114, 104, 26 bp - normal (healthy).

Method Description

The proposed method for the diagnosis of 3-M syndrome in the Yakut population is as follows.

The patient takes blood from the cubital vein into a test tube containing EDTA in the amount of 7-10 ml.

Isolation of DNA from peripheral blood leukocytes is performed by the standard method using the phenol - chloroform method.

For the diagnosis of the 3-M syndrome in the Yakut population (rapid identification of the 4582 insT mutation (Arg 1528 Ser) in the CUL7 gene characteristic of the Yakut population), the necessary DNA fragments are amplified by PCR on a MJ Research RTS-200 programmable thermal cycler in 25 μl of the reaction mixture the following composition: 0.1-1 μg of genomic DNA; 25 pmol of the original oligoprimer (F-GTATGCACTTAGGGCCAGAG; R-CACACTCCTACAGGCTACAC), 200 μM of each nucleoside triphosphate; 0.5 units of DNA polymerase activity (Applied Biosystems), a buffer for the PNR reaction of Applied Biosystems (500 mM KCI, 100 mM Tris-HCI, pH 8.3; 15 mM MCI2; 0.01% gelatin), betaine 1: 2, 20-30 μl of mineral oil. NDP is carried out under the following conditions: initial denaturation - 95 ° - 15 minutes, 35 amplification cycles -: 94 ° - 1 minute, 50-55 ° - 1 minute, 72 ° - 1 minute. Final elongation - 72 ° - 10 minutes. PCR results are evaluated on a 1% agarose gel.

The establishment of a heterozygous carriage of a mutant gene in relatives of a proband is carried out by the method of analysis of polymorphism of lengths of restriction fragments using endonuclease Hinf I (Toyobo). The reaction is carried out according to the protocol of the manufacturer. Amplification results after RFLP analysis are evaluated by electrophoresis in 2% agarose Nusieve gel (drawing).

Normally, fragments of 175, 114, 104, 26 bp should be formed, in the case of carriage in a homozygous state (patient), instead of fragments 114 and 104, fragments 218 bp in length are formed. and 175, 26 bp In the case of heterozygous carriage, mutations (a healthy carrier) of fragment lengths will be 218, 175, 114, 104 and 26 bp.

A quick way to diagnose 3-M syndrome was tested on 39 patients with 3-M syndrome, 35 of their relatives and 20 healthy individuals of Yakut nationality. All patients were homozygotes for this mutation and had alleles 218 bp long. and 175, 26 bp, 30 relatives of 35 were heterozygous carriers of the mutation and had fragment lengths of 218, 175, 114, 104 and 26 bp, the remaining 5 from the group of relatives and 20 healthy fragments were 175, 114, 104, 26 bp Drawing.

This method of diagnosing 3-M syndrome in the Yakut population allows you to make an accurate diagnosis, conduct prenatal DNA diagnostics and prospective medical and genetic counseling for married people. Our proposed method for the diagnosis of 3-M syndrome is simple to perform, accurate and available for execution in the molecular genetic laboratory.

Clinical examples of the disease:

Example 1

Patient K.R., 14 - 09 - 2001 year of birth

Address: Republic of Sakha, Yakutsk, st. Krupskaya 3, apt. 3

Nationality: Sakha

Complaints of short stature, physical developmental lag since birth.

Anamnesis of the disease: For the first time was examined in the neonatal pathology department by geneticists of the medical genetic consultation and diagnosed with Metaphysical chondrodystrophy, type McCusick. Until 2006, the child was observed in the medical-genetic consultation of the Republican Hospital No. 1 - NTC of the Ministry of Health of the Republic of Sakha (Yakutia) with a clinical diagnosis: Metaphysical chondrodystrophy, type McCusick. After molecular genetic analysis, the final diagnosis was made: 3-M syndrome with an autosomal recessive type of inheritance.

Anamnesis of life: A child from 3 pregnancies without a pathology, 2 physiological births in the period of 38 weeks of pregnancy with an increase of 44 cm, weight - 2,980 kg. He screamed after resuscitation, was applied to the chest on the 5th day, transferred on the 5th day to the neonatal pathology unit of Republican Hospital No. 1 - NTC.

Past diseases: SARS, burn disease at 2, 5 years.

Allergic history: calm.

Psychomotor development: keeps the head from 5 months, walks from 12 months. He speaks from 2.5 years.

By pedigree: heredity is not burdened, parents are not relatives, in the family another 1 child is healthy, from his first marriage. Parents are both Saha. Parents' growth is average.

Phenotype

The physique is disproportionate, the head is large in relation to the body. Low growth, dwarfism. The head is hydrocephalic, the face is triangular, large almond-shaped eyes, hypoplasia of the middle third of the face, thick lips, hypotelorism, a long filter. Short nose, cheekbone hypoplasia. Chest deformity of the “cobbler’s chest” type, wide and shortened sternum. The limbs are proportional; the micromelia are upper and lower. Lumbar hyperlordosis. Protruding heels on the feet. Muscle hypotension. Heart tones are rhythmic, clear. The abdomen is large, soft, painless. The liver and spleen are not enlarged. Male type NGOs. Stool and diuresis are normal.

Anthropometric data Date of measurement Age Parameter Value Rating 09-14-2001 0 g 0 m Height (standing), cm 44 Nanism 09-14-2001 0 g 0 m Weight, kg 2,980 Hypotrophy III art. 04/23/2002 0 g 7 m Height (standing), cm 58 Nanism 04/23/2002 0 g 7 m Weight, kg 5,600 Hypotrophy III art. 06-06-2004 2 g 8 m Height (standing), cm 76 Nanism 06-06-2004 2 g 8 m Weight, kg 10 Hypotrophy III art. 04/25/2005 3 g 7 m Height (standing), cm 82 Nanism 04/25/2005 3 g 7 m Weight, kg 10.9 Hypotrophy III art. 04/25/2005 3 g 7 m Head circumference, cm 54 Above the average 03/31/2006 4 g 6 m Height (standing), cm 89 Nanism 03/31/2006 4 g 6 m Weight, kg 12 Hypotrophy III art.

Laboratory test results:

Conclusion No. 21

on molecular genetic research of genomic DNA

On the basis of the Department of Neurology of the Brain Institute of the University of Niigata (Japan), an indirect DNA diagnosis of the following diseases was performed:

1. McCusick metaphysical chondrodystrophy (in the RMPR gene, using polymorphic dinucleotide markers D9S163, 99AC, CA1, D9S1804).

2. Mulibray nanism (at the MUL gene locus using polymorphic dinucleotide markers D17S1606, 95-CA, 272a-CA, 52-CA, D17S924).

3. Laron's disease (in the GH gene using polymorphic dinucleotide markers D5S628, D5S634).

The patient in the loci of the genes of these diseases according to these polymorphic markers is not homozygous, therefore, all these diseases are excluded by the molecular genetic method.

Complete sequencing of the gene Cullin 7 (Cullin7, CUL7). In exon 25 of the Cullin 7 gene (Cullin7, CUL7), which causes a hereditary disease 3-M Syndrome, a nonsense mutation was revealed - insertion of T (thymine) at position 4582. DNA diagnostics was performed by the PNR method followed by RFLP analysis and HinfI restriction enzyme detection the presence of fragments 218 bp long and 175, 26 bp, which is an indicator of the carriage of a mutant gene in a homozygous state (patient).

Conclusion: Thus, according to the results of molecular genetic analysis, the diagnosis of metaphysical chondrodysplasia of McCusick has been removed. As a result of molecular genetic analysis (mutations were found in the CULT gene), the final diagnosis was made: 3 - M syndrome (OMIM-273750).

Functional diagnostics date of Type of study Result 04/25/2002 Rg graph lower limbs Conclusion: no pathology was detected. 12/20/2004 Electroencephalography No pathological activity was detected. 06-06-2005 Electrocardiography Sinus rhythm. Heart rate 115 beats. in min. EOS to the left. 04-04-2006 Ultrasound abdominal Strengthening the vascular pattern of the liver. Compaction of the walls of the CSF of both kidneys (the size of the internal organs corresponds to the height and weight of the child). 04-04-2006 Rg-graphy of the bones of the hand (bone age) X-ray of the visible parts of the bone structure without visible pathology. Specialist consultation date of Specialty Result 03/03/2005 Orthopedist McCusick metaphysical chondrodysplasia. Deformation of the chest 1 step. Flat feet 1 step. 03/03/2005 Endocrinologist Dwarfism against the background of metaphysical chondrodysplasia. 04-04-2005 Optometrist Consultation Moderate retinal angiopathy. 04-04-2005 Pediatrician Metaphyseal chondrodysplasia. 03/27/2006 Speech therapist Conclusion: OHR 2, level 3 against the background of erased dysarthria. 03/27/2006 Orthopedist Conclusion: Deformation of the chest 1 degree. Flat feet 2 degrees. Metaphyseal chondrodysplasia. 03/31/2006 Neurologist Conclusion: REP, hypertension-hydrocephalic syndrome in the stage of subcompensation. Myatonic syndrome. Metaphyseal chondrodysplasia.

Medical report

The diagnosis of metaphysical chondrodysplasia, McCusick type, based on the results of DNA diagnostics, has been removed. Taking into account clinical data (prenatal and postnatal hypoplasia, proportionally low growth, facial dysmorphies, normal intelligence and the absence of endocrine disorders), laboratory and functional studies, molecular genetic analysis (point mutation was identified - insertion of thymine in exon 25 of the CUL7 gene, responsible for development 3 -M syndrome) exposed

FINAL DIAGNOSIS: 3-M syndrome with an autosomal recessive type of inheritance (hereditary nanism (OMIM - 73750). Genetic risk for siblings 25%.

Example 2

Patient M.T.

Date of birth: 27-07-1999

Address: Republic of Sakha (Yakutia), Verkhnevilyuysky ulus, Khoro village.

Complaints of low growth, a sharp lag in growth since birth.

Anamnesis of the disease: The child was examined at the age of 2 weeks in a medical genetic consultation and diagnosed with Metaphysical chondrodystrophy, type McCusick. Until 2006, the child was observed in the medical-genetic consultation of the Republican Hospital - the NTC of the Ministry of Health of the Republic of Sakha (Yakutia) with a clinical diagnosis: Metaphysical chondrodystrophy, type McCusick. After conducting molecular genetic analysis, the final diagnosis was made: 3-M syndrome.

Anamnesis of life: A child from 2 pregnancies in the 2nd half with gestosis. She was sent to inpatient treatment for a period of 37 weeks, by ultrasound of the fetus - intrauterine growth retardation. From 2 operative births at 38 weeks with a weight of 1860, a growth of 35 cm, Apgar 2/6 points. The condition at birth is serious, transferred to the department of pathology of newborns of the Republic of Belarus No. 1-NCM.

Past diseases: SARS.

Allergic history: calm.

Psychomotor development: Holds the head from 5 months, sits - from 8 months, walks independently - from 14 months, first teeth - from 6 months.

From the pedigree: Pregnancy-1 ended with a stillborn fetus of a female, with a weight of 1700 and a hydrocephalic head shape. Sibs with the same phenotype.

Phenotype

The physique is disproportionate, the head is large relative to the trunk, macrocephaly, the trunk is shortened. Short stature, dwarfism. The skin is dark, clean. Head closer to hydrocephalic form. The forehead stands out. The hair is dark. The face is triangular, the nose is simplified. The nose is short with protruding nasal passages. The filter is long. The neck is short. The chest is wide, shortened. Micromelia upper and lower. NPV on both hands. Heart tones are rhythmic, clear, no noise. The abdomen is enlarged, the liver and spleen are not enlarged. Lumbar hyperlordosis. Protruding heels on the feet. Female NGOs. Stool and diuresis are normal

Anthropometric data Date of measurement Age Parameter Value Rating 07/27-1999 0 g 0 m Height (standing), cm 35 Nanism 07/27-1999 0 g 0m Weight, kg 1,860 Hypotrophy 02-09-1999 0 g 2 m Weight, kg 2,100 Hypotrophy 07-08-2000 1 g 1 m Height (standing), cm 57 Nanism 07-08-2000 1 g 1 m Weight, kg 5,400 Hypotrophy 07-08-2000 1 g 1 m Circle gr. C., cm 39 Not acc. Height (standing) 07-08-2000 1 g 1 m Head circumference, cm 44 Below the average 09-09-2004 5 g 1 m Height (standing), cm 83 Nanism 09-09-2004 5 g 1 m Weight, kg eleven Hypotrophy

Laboratory test results:

Conclusion N 23

for molecular genetic research of genomic DNA

On the basis of the Department of Neurology of the Brain Institute of the University of Niigata (Japan), an indirect DNA diagnosis of the following diseases was performed:

1. McCusick metaphysical chondrodystrophy (in the RMPR gene, using polymorphic dinucleotide markers D9S163, 99AC, CA1, D9S1804).

2. Mulibray nanism (at the MUL gene locus using polymorphic dinucleotide markers D17S1606, 95-CA, 272a-CA, 52-CA, D17S924).

3. Laron's disease (in the GH gene using polymorphic dinucleotide markers D5S628, D5S634).

The patient in the loci of the genes of these diseases according to these polymorphic markers is not homozygous, therefore, all these diseases are excluded by the molecular genetic method.

Complete sequencing of the gene Cullin 7 (Cullin7, CUL7). In exon 25 of the Cullin 7 gene (Cullin7, CUL7), which causes a hereditary disease 3-M Syndrome, a nonsense mutation was revealed - insertion of T (thymine) at position 4582. DNA diagnostics was performed by PCR followed by RFLP analysis and Hinfl restriction enzyme revealed the presence of fragments 218 bp in length and 175, 26 bp, which is an indicator of the carriage of a mutant gene in a homozygous state (patient).

Conclusion: Thus, according to the results of molecular genetic analysis, the diagnosis of metaphysical chondrodysplasia of McCusick has been removed. As a result of molecular genetic analysis (mutations were found in the CUL7 gene), the final diagnosis was made: 3-M syndrome (OMIM-273750).

Medical report

The diagnosis of metaphysical chondrodysplasia, McCusick type based on DNA diagnostics is removed. Given the clinical data of the studies (prenatal and postnatal hypoplasia, proportionally low growth, facial dysmorphies, normal intelligence and the absence of endocrine disorders), laboratory-functional and molecular genetic analysis (identified point mutation - insertion of thymine in the 25 exon of the CUL 7 gene, responsible for development 3-M syndrome) exposed

FINAL DIAGNOSIS: 3-M syndrome with an autosomal recessive recessive type of inheritance (hereditary dwarfism (OMIM - 273750). Genetic risk for siblings 25%.

Patient M. D, siblings M.T.

Date of birth: 27-07-1999

Address: Republic of Sakha (Yakutia), Verkhnevilyuysky ulus, village of Khoro,

Complaints of low growth, a sharp lag in growth since birth.

Anamnesis of the disease: The child was examined at the age of 4 months in a medical genetic consultation and was diagnosed with Metaphysical chondrodystrophy, McCusick type. Until 2006, the child was observed in the medical-genetic consultation of the Republican Hospital - the NTC of the Ministry of Health of the Republic of Sakha (Yakutia) with a clinical diagnosis: Metaphysical chondrodystrophy, type McCusick. After conducting molecular genetic analysis, the final diagnosis was made: 3-M syndrome.

Anamnesis of life: A child from 3 pregnancies, proceeding in the 1st half - with toxicosis, in the 2nd half with gestosis. From 3 operative births at 38 weeks with a weight of 1860, an increase of 35 cm, Apgar - 2/6 points. The condition at birth is serious, he was transferred to the neonatal pathology department of the Republic of Belarus No. 1 - NTC.

Past diseases: ARVI

Allergic history: calm.

Psychomotor development: Holds the head from 3 months, sits - from 7 months, walks independently - from 14 months, first teeth - from 6 months.

From the pedigree: Pregnancy - 1 ended with a stillborn female fetus, with a weight of 1700 and a hydrocephalic head shape. Sibs with the same phenotype.

Phenotype

The physique is disproportionate, the head is large relative to the trunk, macrocephaly, the trunk is shortened. Short stature, dwarfism. The skin is dark, clean. Head closer to hydrocephalic form. The forehead stands out. The hair is dark. The face is triangular, the nose is simplified, hypoplasia of the middle part of the face. The nose is short with protruding nasal passages. The filter is long. The neck is short. The chest is wide, shortened. Micromelia upper and lower. NPV on both hands. Protruding heels on the feet. Heart tones are rhythmic, clear, no noise. The abdomen is enlarged, the liver and spleen are not enlarged. Lumbar hyperlordosis. Male type NGOs. Stool and diuresis are normal.

Anthropometric data Date of measurement Age Parameter Value Rating 2003-03-24 1 g 2 m Height (standing), cm 67 Nanism 2003-03-24 1 g 2 m Weight, kg 6.9 Hypotrophy III art. 09-09-2004 2 g 7 m Height (standing), cm 73 Nanism 09-09-2004 2 g 7 m Weight, kg 8.3 Hypotrophy III art.

Laboratory test results:

Conclusion N 24

for molecular genetic research of genomic DNA

On the basis of the Department of Neurology of the Brain Institute of the University of Niigata (Japan), an indirect DNA diagnosis of the following diseases was performed:

1. McCusick metaphysical chondrodystrophy (in the RMPR gene, using polymorphic dinucleotide markers D9S163, 99AC, CA1, D9S1804).

2. Mulibray nanism (at the MUL gene locus using polymorphic dinucleotide markers D17S1606, 95-CA, 272a-CA, 52-CA, D17S924).

3. Laron's disease (in the GH gene using polymorphic dinucleotide markers D5S628, D5S634).

The patient in the loci of the genes of these diseases according to these polymorphic markers is not homozygous, therefore, all these diseases are excluded by the molecular genetic method.

Complete sequencing of the gene Cullin 7 (Cullin7, CUL7). In exon 25 of the Cullin 7 gene (Cullin7, CUL7), which causes a hereditary disease 3-M Syndrome, a nonsense mutation was detected - insertion of T (thymine) at position 4582 / DNA diagnostics was performed by PCR followed by RFLP analysis and Hinfl restrictase enzyme detected the presence of fragments 218 bp long and 175, 26 bp, which is an indicator of the carriage of a mutant gene in a homozygous state (patient).

Conclusion: Thus, according to the results of molecular genetic analysis, the diagnosis of metaphysical chondrodysplasia of McCusick has been removed. As a result of molecular genetic analysis (mutations found in the CUL7 gene), the final diagnosis was made: 3 M syndrome (OMIM-273750).

Functional diagnostics date of Type of study Result 03-03-2003 Rg graph lower limbs Conclusion: metaphysical chondrodysplasia, type McCusick, is not ruled out. 09/09/2004 Skeleton Rg Conclusion: Indirect Signs of I / H Hypertension? 09/09/2004 Rg-graphy of the bones of the hand (bone age) Conclusion Corresponds to 12-15 years. 09-09-2004 Ultrasound abdominal Sealing the walls of the CLS of both kidneys. 09-09-2004 Thyroid ultrasound Pathology is not revealed. 09-09-2004 Ultrasound of the prostate Echoes of testicular hypoplasia. 09-09-2004 Electrocardiography Rhythm - moderate sinus, such a cardiac heart rate of 136 beats / min. The vertical position of the EOS. Signs of left atrial hypertrophy. Severe violations of the repolarization processes as dystrophic changes (combine with the clinic). ECG - control. 09-09-2004 Rg-graphy of the lumbar spine Conclusion: Pathology was not detected. 09-09-2004 Doppler echocardiography Additional trabecula in the LV cavity. The cavity system of the heart is not expanded. LV systolic function retained PV 65% 09-09-2004 Electroencephalography Bea goal brain by age. Minor cerebral changes of an irritative nature. Focal, epigastric nget. Specialist consultation date of Specialty Result 03-03-2003 Neurologist Diagnosis: hydrocephalic syndrome. 03-03-2003 Orthopedist Given the radiological data, it is recommended that an orthopedic surgeon is monitored at least once every six months. 09-09-2004 Neurologist Hydrocephalic cm. The condition is compensated, does not need treatment. 09-09-2004 Optometrist Consultation DZ: Angioretinopathy OI, 09-09-2004 Orthopedist The diagnosis at this time does not contradict the diagnosis: Metaphyseal dysplasia. Recommended: - dynamic observation; - exercise therapy, massage.

Medical report

The diagnosis of metaphysical chondrodysplasia, McCusick's type, based on DNA diagnostics, has been made. Based on clinical data (prenatal and postnatal hypoplasia, proportionally low growth, facial dysmorphies, normal intelligence and the absence of endocrine disorders), laboratory and functional studies and molecular genetic analysis (identified point mutation - insertion of thymine in exon 25 of the CUL7 gene, responsible for the development of 3-M syndrome) FINAL DIAGNOSIS is set: 3-M syndrome with an autosomal recessive inheritance type Nia, (hereditary dwarfism (OMIM -. 273750) Genetic risk to the sibs of 25%.

Claims (1)

A method for the diagnosis of 3-M syndrome by molecular genetic analysis by PCR, characterized in that in the diagnosis of 3-M syndrome in the Yakut population with an autosomal recessive type of inheritance using F-GTATGCACTTAGGGCCAGAG primers; R-CACACTCCTACAGGCTACAC carry out amplification of genomic DNA isolated from peripheral blood leukocytes and analysis of restriction fragment length polymorphism using Hinfl endonuclease, and in the presence of 218 bp fragments and 175, 26 bp diagnose the carriage of the mutant gene Cullin 7 (CUL7) with a mutation of 4582 insT (Arg 1528 Ser) in the homozygous state (patient), in the presence of fragments -218, 175, 114, 104 and 26 bp diagnose carriage of a mutant gene in a heterozygous state and with a fragment length of 175, 114, 104, 26 bp diagnose a normal gene (healthy).

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