Prenatal Diagnosis of LSDs

Introduction
Increased awareness, investigational facilities and available treatment option for various lysosomal storage disorders (LSDs) has generated the great deal of interest and hope to prevent and cure for this group of diseases. Postnatal confirmation and Prenatal diagnosis (PD) is one of the important parts in counseling to the affected families for prevention of the disease with treatment option are either not available or beyond reach to the family. Various methods have been known and employed in the prenatal diagnosis of LSDs that include enzyme study from uncultured chorionic villus cells (CV), morphological study of CV sample for vacuoles, cultured chronic villus (CT) and/or amniotic fluid (AF) cells, electrophoresis of amniotic fluid for excretion pattern of various mucopolysaccharides, electroscopic ionization tandem mass spectrometry (Ramsay S et al., 2004) and mutation analysis using DNA from uncultured chorionic villous or amniotic fluid cells.

Though molecular study provides specific diagnosis of the LSDs, identification of the known mutation in an affected child is the prerequisite for confirmed prenatal diagnosis. This require database of common mutation observed in Indian population which is not known except for Ashkenazi Jewish population. Therefore, in most part of the world lysosomal enzyme study is prerequisite for prenatal diagnosis followed by mutation study as a confirmation where mutation is known. Present study has been undertaken with a view to establish the prenatal diagnosis of LSDs using enzyme study from CV, CT or AF and demonstrate the specialty of reliability of enzyme study in the prenatal diagnosis of LSDs.   
 
Patient selection
Prospective study of 28 pregnancies from 2001 to 2008 were selected for the PD either by chorionic villus or Amniotic fluid study for Lysosomal enzymes were index case diagnosis was confirmed. Prior to written informed consent was obtained as per prenatal diagnosis act; counseling was provided to each family.

TABLE demonstrates the normal range of lysosomal enzymes in various tissues in pregnant women who delivered normal child and subsequent follow-up was done till two years of age confirming the normal status.

Out of 28 pregnancies, preponderance was observed for MPS (25.53%); NPD-A & B (21%), GM-2 Gangliosidosis (17%) and Gaucher’s disease (12.77%). Present study included 28 pregnancies at risk for LSDs. They were NPD type A & B, Gaucher’s disease, GM-2 gangliosidosis, Pompe disease, MPS-I (Hurler disease), MPS-III B (Sanflippo disease), MPS-IV-B (Morquio B), MPS-VI (Maroteaux–Lamy disease), MPS-VII (Sly syndrome), and Metachromatic Leucodystrophy (MLD). Of these, five pregnancies (17.85%) were found to be affected with various LSDs with enzyme activity of less than 10% except one of MPS-IVB with high residual activity, five (17.85%) have shown the carrier status with 50% enzyme activity expression while eighteen (64.3%) have shown normal enzyme activities (TABLE-II). Due to high residual activity of b-galactosidase in suspected case of MPS-IVB further study was carried out from AF by electrophoresis of glycosaminoglycans (GAG) excretion pattern and enzyme study from cultured AF cells. GAG excretion was found in excess of keratin sulfate with nearly 5% activity of enzyme confirming affected status of the fetus.

In all five affected pregnancies; confirmation of PD was made by Lysosomal enzymes study from abortus fibroblasts and all have shown very low to undetectable activity of the enzymes reconfirming the PD. Twenty three pregnancies with normal or carrier status in PD were followed up till one year of child age and selective study by Lysosomal enzyme was found to be normal reconfirming the PD except in two case. This includes one; where b–glucosidase activity was normal in CV and CT cells, the child was found to have NPD-A after delivery with significantly low activity of sphingomyelinase. While in another case, CT cells were investigated for Hex-A and T and post-delivered child was found to have b-Thalassemia major. In both of these cases, parental carrier status showed, confirmed heterozygous for two different disorders: NPD-A/Gaucher in one and Hex-A and b-Thalassemia in another.

 

Deficient Enzyme	No of Sample studied	Enzyme activity in nmol/hr/mg protein
		Uncultured CV	Cultured CV	Cultured Amniotic fluid cells
Sphingomyelinase	4	22.2±9.9	51.6±43.7	11.25±0.78
Hexosaminidase [A] Hexosaminidase [Total]	5	28-63% 1908±900	21-63% 2247±643	40-65% 1417.7±243.51
b-Galactosidase	5	170±55.8	350±147	351.94±121.22
b-Glucosidase	2	175±101	156±77.1	169.75±55.13
µ-Iduronidase	2	8.52±4.47	ND	49.6±6.84
N-Ac-a-glucosaminidase in plasma	1	53.47	42.76	26±8.7
Arylsalfatase B	2	323±89	212±106.7	46.68±5.21
b-Glucuronidase	2	102±38.7	31.8±14.8	56.97±13.51
Arylsalfatase-A	4	123±61.4	109±60.4	97.81±42.2
µ-1-4-glucosidase	1	77.4±5.67	ND	82.16±28.19