PRENATAL DIAGNOSTICS OF MUCOPOLYSACCHARIDOSIS LYSOSOMAL STORAGE DISEASE

GENETIC CASE STUDY OF SANFILIPPO SYNDROME PATIENT

Alizada Sevda Aydin

Docent, PhD on Biology, Azerbaijan Medical University,

Azerbaijan, Baku

Elkhan Mammad Rasul Rasulov

Professor, SciDr (Biology - Human genetics), Genom clinic laboratory,

Azerbaijan, Baku

Sanfilippo syndrome (Mucopolysaccharidosis type III) being a lysosomal storage disease, causes polyorgan deficiency because of heparansulfate (HS) storage in cells and tissues. Sanfilippo syndrome was described by doctor Silvester Sanfilippo in 1963, and he was the first who studied the clinics. The early manifestations could be seen as cognitive changes, neurologic disorders, speech delay (loss), regression in gained skills, hyperactivity, autistic spector, insomnia, epylepsy, aggresiveness, movement difficulty, hepatomegalia [6,8].

Sanfilippo syndrome has A, B, C and D genetic forms with very similar clinic manifestations. Each form gene locates in different chromosomes. Sanfilippo type A gene locates on the long shoulder of the 17th cromosome (17q25) and provides synthesis of N-sulfoglucosamine sulfohydrolase enzyme (SGSH: 605270). N-alfa-acetylglucosaminidase (NAGLU:609701) enzyme gene responsible for Sanfilippo type B locates on the long shoulder of chromosome 17 (17q21). HGSNAT gene (610453) in Sanfilippo syndrome type C locates on the short shoulder of chromosome 8 and pariticipates in synthesis of Heparane acetyl-CoA-glucosaminide-N-acetyltransferase. Gene for Sanfilippo syndrome type D locates on the long shoulder of chromosome 12 (12q14) and assures synthesis of N-acetylglucosamine-6-sulfatase enzyme (607664). Inheritance type is autosome recessive [4,7].

It should be mentioned that problems linked with lysosome storage diseases are not studied for children-patients in Azerbaijan Republic. Data of clinic manifestations for lysosome is completely absent. There are our reference data published on genetic studies of the said Mucopolysaccharidoses cases in Azerbaijan. This study is the continuation of these researches [1-3].

It should be also mentioned that there is high frequency of beta-thalassemia carriage in Azerbaijan. According to data of World Health Organization the highest frequency of among the countries in the world is observed in the countries of Mediterranean coasts such as Sardinia island (Italy) 23-30%, Cyprus island - 17%, Greece - 10-12%, Turkey - 3-12%, etc. It is around 10 years that pre-marriage screening of couples is foreseen to find out couples with risk of beta-thalassemia. This screening is designed to reveal those couples for further fetal prenatal diagnostics during pregnancies. Data of this screening says that 7,6% of our young people are carriers of beta-thalassemia. But number of heterozygotes on beta-thalassemia in some regions of Republic reaches 10% and even higher.

Goal of our studies is identification and genetic study of Mucoplysaccharidoses type III (Sanfilippo syndrome) using modern biochemical and molecular diagnostics methods and techniques. Taking into considerations high acquaintance of beta-thalassemia in the population in our Republic, we put up the task to identify and study genetics of beta-globin gene (HBB).

The patient is found during medical-genetic consultancy in one of medical centers in Baku city, Azerbaijan. Medical-genetic consultancy was conducted in front of doctor-pediatrician and doctor-geneticist. Clinical manifestations corresponded to Mucopolysaccharidosis type III (Sanfilippo syndrome).

Patient suffers this disease since early childhood as “specific behavioral disturbances”: Mental retardation, no contacts with society, aggressiveness and speech disorders as well as coarse facial features. In girls we see often hirsutism, but mentally good development. Normally there are no skeletal disorders. Patients have got storage one of kinds of glycosaminoglycans - heparansulfate - in neural system cells and urinary excretion that leads to progressive mental retardation.

To diagnose Sanfilippo syndrome we used urine and capillary blood absorbed and dried in DBS (Dry Blood Spot) cards as well as venous blood in 2ml. Beta-thalassemia diagnostics was carried out with electrophoresis of hemolysate prepared from venous blood at the same time evaluating fetal hemoglobin. Results of hemoglobin electrophoresis with the following quantitative evaluation of HbA₂ fraction showed its increase to 4,3% when norm was 3,5% of total hemoglobin. Level of fetal hemoglobin (HbF) was increased as well - 4,3% (norm - 2,5%). Hence, minor hemoglobin fraction indications (HbA_2, HbF) witness of patient as a heterozygous carrier of bet-thalassemia.

Considering that the basics of all Mucopolysaccharidoses is enzyme activity disorder, we started firstly from the identification of enzyme levels specific for every of four types of Sanfilippo syndrome (A, B, C and D). The following enzymes were used: Heparane-N-sulfatase (Sanfilippo А), alfa-N-acetyl-D-glucoaminidase (Sanfilippo В), Heparane acetyl-CoA-glucosaminide-N-acetyltransferase (Sanfilippo C) and N-acetylglucosamine-6-sulfatase enzyme (Sanfilippo D). For all four patients total enzyme deficiency was found out. Total Heparane acetyl-CoA-glucosaminide-N-acetyltransferase enzyme activity deficit was identified for the patient and it is characteristic of Sanfilippo Type C. Here we observed total enzyme deficiency with zero activity (0,0(LOD)μmol/L/h) which is characteristic of homozygous or double heterozygous (compound) state when the norm should be ≥2,0 µmol/L/h. Genealogical analysis allowed us to find that patient’s parents were relatives - cousin marriage type.

Enzyme analysis of index patient’s parents showed the following case: parents revealed enzyme deficit corresponding to heterozygous carriers.

DNA obtained from patient’s peripheral blood sample was studied with NGS (New Generation Sequencing) technique. A custom double stranded DNA capture bait pool was used to selectively enrich the coding regions, 10 bp of flanking intronic sequences, and known relevant variants beyond the coding regions, based on HGMD^® and CentoMD^® for the 166 panel genes. Libraries are generated with Illumina compatible adaptors, and sequenced on an Illumina platform to obtain ≥ 50x coverage depth for >99,5% of the targeted bases. Mean depth of reading consists of 1559 indications. All potential disease-causing variants, including the ones reported in HGMD^®, in ClinVar and in CentoMD^® are considered. The investigation for relevant variants is focused on coding exons and flanking +/-10 intronic bases. All potential modes of inheritance patterns are considered. Centogene^® has established stringent quality criteria and validation processes for variants detected by NGS. Pathogeny classification of the obtained results was considered according to «Guidelines of ACMG*». At the same time sequencing of SGSH gene was carried out [5].

To diagnose primerily we analysed quantitively and qaulitevely for levels of glycosaminglycans (GAG) in urine with thin layer chromatography technique. Being a precise diagnostic method we analysed blood serum, leucocytes, fibroblasts to evaluate corresponding enzymes’ activities. Genetic analysis on DNA level makes diagnose more precise.

14-year-old patieint (male) was identified during clinical examination with doctor-pediatrician and doctor-geneticist. To identify Sanfilippo syndrome and its type enzyme activities of four different genes were analysed.

Mass-spectrometry technique was used for evaluation of enzyme activities for all fourtypes of Sanfilippo syndrome. We got total deficiency of only one enzyme: Heparane acetyl-CoA-glucosaminide-N-acetyltransferase. This state is characteristic for Sanfilippo syndrome type C. Molecular analysis of HGSNAT gene (NM_152419) was conducted with NGS (Next generation sequencing) technique. The result of genetic analysis showed us G nucleotide dupplication in position 1345 of exon 13 in homozygous state (c.1345dupG). As a result of mutation there occur a reading frame shift and the normal codon 21 is substituted with stop codon. Consequently, there happens a change of Asparagine in normal protein with Glycine in 449 position caused with the saidabove mutation (Asp449Glyfs*21).

This mutation was described earlier as disorder causing MPS III. In ClinVar ^® list this mutation is stated as pathogenic. Clinic testing identified a variant with ID: 1231. It is classified as pathogenic (class 1) in accordance with Recommendations of Centogene^® and ACMG^® [5].

Epidemiologic researches in West Australia during 1969-1998 revealed 11 cases with MPS III (1:58 000), 5 patients in MPS IIIA and MPS IIIB each, one patient with identified MPS IIIC [8].

Кhаn et al., 2017, examining 467 patients from Japan and Switzerland during 1982-2009, all types of MPSs we diagnosed. Frequency counted as 1,53 per 100 000 live newborns. MPS types were distributed as follows: MPS II was 55% of all patients with MPS (0,84:100 000), frequencies for MPS I, MPS III, MPS IV, MPS VI and MPS VII - 15%, 16%, 10%, 1,7% and 1,3% relatively.

In Japan frequency was 1,43 per 100 000 live healthy newborns. Sanfilippo syndrome was counted as 16% from all types of disease right next to MPS II (55%). In Switzerland the frequency of MPS was 1,56 per 100 000 live healthy newborns, where MPS III frequency 24% of all patients [6].

Prevalence of Sanfilippo syndrome in different countries in the world is a way variable: about 1 per 280 000 born in Northern Ireland, up to 1 per 66 000 in Australia and 1 per 50 000 in Netherlands. Sanfilippo type A mainly prevails in North-West Europe, type B - in South-East Europe, types C and D are much rarer [6-8].

Thus, for the first time medical-genetic study of affected children to diagnose Sanfilippo syndrome - lysosomal storage disease - was carried out in Azerbaijan Republic. In one of patients we managed to identify and describe Sanfilippo syndrome according to clinical manifestations. To confirm provisional diagnosis on the basis of clinical manifestations, biological material as urine and blood samples for biochemical and genetic analysis was used. Results of carried out biochemical and genetic analysis allowed us diagnose the disease as well as identify a mutation type.

We managed identifying additional HBB gene mutation in heterozygous state. Substitution of Cytosine nucleotide with Guanine nucleotide happened in the position 745 of intron 2 in HBB gene (IVS II-745 C>G). This mutation was described for Middle East and Asia countries.

Hence, we found out total deficiency of Heparane acetyl-CoA-glucosaminide-N-acetyltransferase and identified  HGSNAT gene mutation c.1345dupG: Asp449Glyfs*21.

Taking into account reproductive age of parents, we offered them fetus prenatal diagnostics when next pregnancy.

References:

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