Spectrum of Lysosomal Storage Disorders at Tertiary Centre: Retrospective Case-Record Analysis

Abstract

Lysosomal storage disorders (LSDs) are relatively common slow progressive inborn error of metabolism encountered by clinicians. This work intends to highlight the more common LSDs, their clinical presentation, outcome, and mutation (wherever feasible) collected from the genetic clinic at tertiary care center in Eastern Uttar Pradesh. The data for analysis were collected retrospectively from genetic records from a follow-up clinic. All cases < 18 years of age were analyzed. Cases with LSDs with confirmed enzyme results were enrolled in this study. Clinical profile, screening test results, and outcome were collected. There were 32 cases including 27 males and 5 females in this cohort: 8 Gaucher disease (GD) patient and 24 non-GD patients. GD (type 1) is the commonest LSD in GD group. Anemia, thrombocytopenia, splenomegaly, and hepatomegaly were the consistent finding in patients with GD (type 1). L483P mutation was reported in two GD patients. One GD patient is on enzyme replacement therapy for 2 years and is currently doing well. The commonest disorders in non-GD were mucopolysaccharidosis (MPS) ( n  = 11), metachromatic leukodystrophy ( n  = 4), I-cell disease ( n  = 3), Niemann-Pick A/B ( n  = 3). MPS-II is the commonest MPS among non-GD group.

Introduction

Lysosomal storage disorders (LSDs) are large molecule inborn errors of metabolism that result in accumulation of carbohydrates, proteins, fats, and nucleic acid within the cell. ¹ LSDs usually occur due to deficiency of lysosomal enzymes but can also result from deficiency of enzyme trafficking protein, membrane proteins, and activator proteins. ² To date, there are 50 different LSDs causing 40 different storage disorders. ^{3 4} Individually, they are rare but collectively they have a frequency of 1 in 5,000 live births worldwide. ⁵ Few of LSDs are now treatable with enzyme replacement therapy (ERT); therefore, early identification through their clinical presentations by pediatricians is the key to achieve a better outcome. There are only a few studies highlighting the spectrum of LSDs at genetic clinics and diagnostic laboratories. ^{6 7 8 9} These studies are centered on metropolitan cities in India. The data of number of cases, clinical spectrum, and their outcome from small cities and region around Eastern Uttar Pradesh were yet to be published.

Methodology

This study was a retrospective record analysis of 32 LSD patients from 29 unrelated families, who have visited the genetic clinic of the Department of Pediatrics in a period from February 2014 to May 2019. Institute's ethical clearance was obtained. Data were collected after taking informed consent from parents or primary caregivers. All relevant demographic, clinical records, investigations, radiographs, photos, mutation profile were documented in predesigned performa. The clinical history including disease onset, family history, affected family member(s), history of sibling death, and consanguinity was carefully attained. Examination included anthropometry measurement (weight, height/length, upper segment, lower segment, US/LS ratio, head circumference, arm span), dysmorphology assessment (by two independent dysmorphology experts), and systemic examination as per standard protocol including fundus examination. Investigations were further performed based on each case. Screening investigations included chitotriosidase (for storage disorders), p-pyrocatechol sulphate for I-cell disease, toluidine blue spot test for mucopolysaccharidosis (MPS), skeletal survey for Morquio disease, neuroimaging for leukodystrophies, fundus exam for cherry-red spot, and bone marrow examination (for suspected storage disorders), which were followed by definitive enzyme analysis on peripheral leucocytes or dried blood spot and mutation analysis (wherever feasible)

Results

This cohort consisted of 27 males and 5 females from a total of 29 different families with the presence of third-degree consanguinity in seven families (two in GD group vs. 5 in non-GD group). The average age, median age, and modal age when the patients were evaluated at our hospital were 44, 38, and 60 months, respectively. Age ranged from 2 to 126 months. The mean age of presentation of GD was 40 months (six males and two females from eight different families). Youngest patient of GD was 2 months old and the oldest patient was 96 months old at the time of diagnosis. All patients with GD presented with anemia, thrombocytopenia splenohepatomegaly ( Table 1 ). The average size of hepatomegaly was 6.62 ± 2.37 cm below the costal margin. The average size of spleen enlargement was 12.37 ± 2.59 cm below the costal margin. Mean hemoglobin (Hb) value of patients with GD was 7.01 ± 1.5 g/dL. Mean total leucocyte count of patients with GD was 5,537.5 ± 865.36 cells/cmm. Mean platelet count at admission for GD was 74,250 ± 43,677; mean enzyme (β-glucocerebrosidase activity) was 0.98 ± 0.336 nmol/h/mg. Molecular studies were performed in only two patients with GD (GD 5 and GD 6), which revealed L483P pathogenic mutation. There were two deaths in Gaucher disease (GD) patients in the hospital. Three GD patients are on supportive treatment. Two patients were lost to follow-up. One GD patient is on ERT since May 2017. The parameters at time of enrolment for therapy at age of 6 years were weight: 16 kg; height: 110 cm; Hb: 8.1 g/dL; total leucocyte count: 4,900/µL; platelet count: 44,000/µL; liver span: 14 cm; spleen: 16 cm. The post-therapy parameters at age of 8 years were weight: 20 kg; height: 120 cm; Hb: 10.2 g/dL; total leucocyte count: 6,420/µL; platelet count: 110,000/µL; liver span: 6 cm; spleen: 4 cm. The average age of presentation in non-GD group was 46 months ( Table 2 ). It comprised of 24 patients from 22 different families (21 males and 3 females). There were five families with third-degree consanguinity. Among non-GD group, patients of mucopolysaccharidoses (MPS-I, n  = 3; MPS-II, n  = 6; MPSIVA, n  = 2) were most frequently encountered. Six patients from four different families had MPS-II. Two siblings each from two different families were affected. Other patients included I-cell disease ( n  = 3), metachromatic leukodystrophy ( n  = 4), Pompe disease ( n  = 2), Niemann–Pick disease A/B ( n  = 3), and Sandhoff disease ( n  = 1). All non-GD group patients were provided with supportive treatment and genetic counselling for the evaluation of the risk of recurrence.

Table 1. Clinical, biochemical, mutational, outcome profile of Gaucher disease patients ( n  = 8) .

S.No.	Problem at presentation	Locality	Age at presentation	Gender	Organomegaly	Investigation	Enzyme level (β-glucocerebrosidase activity); n  > (4 nmol/h/mg)	Outcome
GD1	Abdominal distension for 25 d Fever for 10 d	Rohtas (Bihar)	7 mo	Male	Liver: 5 cm; spleen: 16 cm	Hb: 5.7 g/dL, TLC: 6,100/cmm DLC: N30L60E1M9 Platelet count: 2,000/cmm	0.67	Death at day 7 of admission
GD2	Fever on and off for 1 y Progressive distension of abdomen for 1 y	Buxar (Bihar)	8 y	Male	Liver: 6 cm; spleen: 13 cm	Hb: 9.6 g/dL, TLC: 4200/cmm, platelet count: 65,000/cmm	1.20	Lost to follow-up
GD3	Abdominal distension for 3 mo Pain abdomen: 3 mo Fever: 15 d	Azamgarh (UP)	5 y	Male	Liver: 8 cm; spleen: 15 cm	Hb: 6.5 g/dL, TLC: 6700/cmm, DLC: N18L79M1E2, platelet count: 70,000/cmm	1.8	Under ERT since May 2017
GD4	On and off fever for 1 y Loose stools for 1 mo	Not available	5 y	Male	Liver: 1 cm; spleen: 8 cm	Hb: 7.5 g /dL, TLC: 3,790/cmm, DLC: N40L40M6E6, platelet count: 1.7 lakhs/cmm	0.50 nmol/hr/mg	Lost to follow-up
GD5	Abdominal distension for 5–6 mo	Deoria (UP)	3 y, 6 mo	Female	Liver: 5 cm; spleen: 10 cm	Hb: 10 g/dL, TLC: 5,580/cmm, DLC: N43L50M4E2, platelet count: 1.04 lakhs/cmm	0.53	Supportive treatment, applied for government aid
GD6	On and off vomiting	Gopalganj (UP)	3 y	Male	Liver: 13 cm; spleen: 18 cm	Hb: 8.2 g/dL, TLC: 6,800, DLC: N46L46, platelet count: 1.5 lakhs/cmm	0.54	Supportive treatment, applied for government aid
GD7	Fever for 20 d Cough for 10 d Vomiting for 4 d	Aurangabad (Bihar)	15 mo	Female	Liver 7 cm; spleen: 11 cm	Hb: 3.5 g/dL, TLC, 4,380/cmm, DLC: N49L33, platelet count: 20,000/cmm	1.3	Supportive treatment
GD8	Fever for 20 d Paleness of body for 20 d	Gazipur (UP)	2 mo	Male	Liver: 8 cm; spleen: 8 cm	Hb: 5.1 g/dL, TLC: 6,750/cmm, DLC: N23L69M8, platelet count: 13,000/cmm	1.3	Death

Abbreviations: cmm, cubic mm; DLC, differential leukocyte count; Hb, hemoglobin; TLC, total leucocyte count.

Table 2. Profile of non-Gaucher disease patients ( n  = 24, families: 22) .

Type of LSD	Number	Enzyme level	Any comment
Mucopolysaccharidosis-I	3 (1 m/2 f)	P1: 3.1 nmol/h/mg	No consanguinity, all three patients from three different families
		P2: 3.1 nmol/h/mg
		P3: 2.5 nmol/h/mg
Mucopolysaccharidosis-II	6 (m)	P4:0.058 nmol/4h/mg	No consanguinity, P4, P5 were brothers; P6, P7 were brothers; total six patients from four different families
		P5: 0.062 nmol/4h/mg
		P6: 0.09 nmol/4h/mg
		P7: 0.062 nmol/4h/mg
		P8: 0.00 nmol/4 h/mL
		P9: 0.00 nmol/4 h/mL
Mucopolysaccharidosis-IV A	2 (m)	P10:0.04nmol/17h/mg protein	No consanguinity, all two patients from three different families.
		P11: 0.06 nmol/17 h/mg protein
Metachromatic leukodystrophy	4 (m)	P12: 5.92 nmol/h/mL	No consanguinity, all four patients from four different families
		P13: 4.16 nmol/h/mL
		P14: 3.42 nmol/h/mL
		P15: 5.92 nmol/h/mL
I cell disease	3 (m)	P16: Arylsulfatase A; hexosaminidase (total); β-glucuronidase levels high	Three different patients from three different families with presence of third-degree consanguinity in each family
		P17: Arylsulfatase A; Hexosaminidase (total); β-glucuronidase levels high
		P18: Arylsulfatase A; hexosaminidase (total); β-glucuronidase levels high
Niemann pick (A/ B)	3 (m)	P19: 0.78 nmol/17h/mg protein	Three different patients from three different families with third degree consanguinity present in one family
		P20: 0.53 nmol/17h/mg protein
		P21: 0.42 nmol/17h/mg protein
Pompe disease	2 (m)	P22: Ratio of lysosomal α-glucosidase to total α-glucosidase is 0.19	Two brothers from one consanguineous family
		P23: Ratio of lysosomal α-glucosidase to total α-glucosidase is 0.193
Sandhoff disease	1 (m)	P24: Total hexosaminidase (A+ B)–< 0.062 nmol/h/mL	One patient from nonconsanguineous family

Abbreviation: LSD, lysosomal storage disorder.

Discussion

LSDs are relatively common in the pediatric age group (<18 years). Table 3 depicts the frequency of various LSDs at various genetic clinics and diagnostic laboratories across the country. GD and MPS constitute the most common disorders at all centers in our country ( Table 3 ). I-cell disease (mucolipidosis II/III), Farber disease, Pompe disease, and Fabry diseases are among the least common LSD presenting to genetic clinics. Diagnostic laboratories have a greater number of cases as compared with genetic clinics due to the increased number of referrals to these centers for diagnosis. GD is the most common LSD presenting to our genetic clinic, as GD is the most common reported LSD worldwide and in India. ^{9 10} All eight patients of GD had anemia, thrombocytopenia, splenomegaly, and hepatomegaly without neurological involvement. The molecular study was performed in two patients. It was L483P (previously denoted as L444P). L483P is the commonest mutation reported in Indian patient of GD and worldwide. ^{11 12 13} Studies suggested that L483P mutation was associated with the neuronopathic type of GD (type 2 and 3). ^{14 15 16} On the contrary, Sheth et al have found L483P mutation in all the three types of GD, more commonly associated with type 1 GD. ¹³ We also found L483P mutation in our two type 1 GD patients. This phenotypic variation in different population due to L483P mutant allele has been explained by the effect of the modifier gene on the mutant allele. Many of our GD type 1 patients may develop neurological symptoms at a later stage of the disease. One GD patient was put on ERT through the charitable program. The child has achieved all therapeutic goals set for ERT. ¹⁷ There is an increase in weight, height, Hb by 2.1 g/dL and an increase in platelet count by 56,000/µL, decrease in liver and spleen span by more than 50%. Very few centers in India are equipped to treat GD. They are Bengaluru (Indira Gandhi Institute of Child Health and Center for Human Genetics), Chennai (Fetal Care Research Foundation), Hyderabad (Rainbow Children's Hospital), Kochi (Amrita Institute of Medical Sciences), Lucknow (Sanjay Gandhi Postgraduate Institute of Medical Sciences), Mumbai (KEM Hospital and Jaslok Hospital), and New Delhi (AIIMS and Sir Ganga Ram Hospital). Till 2017, 105 GD patients are receiving ERT through various charitable programs of ERT producing companies (Sanofi-Genzyme, Shire, Protalix). ¹⁸ Few patients (exact number not known) are receiving ERT through central and state government. So, it is the need of time to collaborate with all centers in India to know the exact short- and long-term outcome of Indian Gaucher patients.

Table 3. Types of lysosomal storage disorders diagnosed at various genetic clinics and diagnostic laboratories across India.

Parameter	Agarwal et al 6	Verma et al 7	Kadali et al 8	Sheth et al 9	Present study
Period of study	Jan 2002–Dec 2013	Jan 2008–Dec 2010	Nov 2007–May 2012	Jan 2002–December 2012	Feb 2014–August 2019
Site of study	Genetic Clinic, Department of Pediatrics, KEM Hospital, Mumbai	Department of Medical Genetics SGPGI, Lucknow	Sandor Lifescience Private Ltd, Hyderabad, Medical Genetics Laboratory	Department of Biochemical and Molecular Genetics, FRIGE, Ahmedabad	Genetic Clinic, Department of Pediatrics, IMS-BHU
Region of study	Mumbai and around	North India	Pan India	Mostly from Western India	Eastern Uttar Pradesh
Age range	3.5–274 mo	5 mo–26 y	0–16 y	1 d–16 y	2–126 mo
Sphingolipidoses
Gaucher disease	38	10	41	60	8
GM1 gangliosidosis	10	6	28	30	–
Tay–Sachs disease	9	4	24	39	–
Sandhoff disease	9	3	11	30	1
Niemann–Pick disease	9	3	12	24	3
Metachromatic leukodystrophy	8	10	29	38	4
Krabbe disease	5	–	34	17	–
Fabry disease	–	2	3	–	–
Farber disease	1	–	–	–	1 suspected but not confirmed
Mucopolysaccharidoses (MPS)	24	23	102	85	11(MPSII-6; MPSI-3; MPSIVA-2)
Neuronal ceroid lipofuscinosis	3	1	25	18	1 suspected
Mucolipidosis II/III	2	3	11	11	3
Pompe disease	1	3	47	19	2
Wolman disease	–	1	2	–	–

The non-GD patient group comprised of 24 patients, most of them being with MPS (11/24), specifically type 2 MPS ( n  = 6). Coarse facies, umbilical hernia, short stature, and multiple dysostosis complex on radiographs were the consistent features in all six patients. One patient presented with pebbling of skin as a cutaneous marker of Hunter syndrome ( Fig. 1 ), which has been previously reported in the literature. ^{19 20 21 22} Three cases of I-cell diseases (mucolipidosis II/III) presented with coarse facies, short stature, gingival hyperplasia, multiple dysostosis complex on radiographs, and negative toluidine blue test. In one of I-cell diseases, we could identify the disease-causing mutation that has been published. ²³ These three cases highlight the importance of suspecting I-cell diseases in the presence of MPS-like features with negative toluidine blue dye test. Although we could diagnose few treatable LSD such as GD, MPS 1, MPS 2, Pompe disease, and MPS IVA (19/32), we could offer definitive treatment to only one patient with GD. All the remaining patients were given supportive treatment. Genetic risk reoccurrence in future pregnancies and screening of family members were offered to families. Lifelong cost of treatment (ERT) cannot be borne by patients as they are from very poor strata. Besides this, there is no insurance cover for genetic diseases in India at present. Here comes the role of government agencies and philanthropy organizations to support research and create technology to prepare enzyme indigenously to reduce the cost of ERT. The governments should come up with orphan drug policy, negotiate patent transfer, and establish its own laboratories to produce such drugs in large quantity to resolve this problem in India or other developing countries.

Fig. 1.

Pebbling of skin in one of mucopolysaccharidoses II patient.

LSDs are important group of inherited disorders that present to any genetic clinic. However, the lack of funding, long-term follow-up and genotype testing for all LSDs are the challenges. Early identification through their clinical presentations by pediatricians is the key to achieve a better outcome.