Clinical Characterization of Mucolipidoses II and III: A Multicenter Study

Abstract

Mucolipidoses (MLs) II and III are rare lysosomal diseases caused by deficiency of GlcNAc-1-phosphotransferase, and clinical manifestations are multisystemic. Clinical and demographic data from 1983 to 2013 were obtained retrospectively. Twenty-seven patients were included (ML II = 15, ML III α/beta = 9, ML III gamma = 3). The median age at diagnosis was 2.7 years. The predominant clinical presentations were skeletal symptoms. The ML II patients showed physical and cognitive impairment, while the ML III α/beta patients have more somatic abnormalities and usually were delayed in early development as compared with ML III gamma patients. This is the most comprehensive study exploring characteristics of Brazilian patients with MLs II and III.

Introduction

Mucolipidosis (ML) II, ML III α/beta, and ML III gamma (OMIM #252500, #252600, and #252605) are rare, autosomal recessive genetic diseases caused by deficiency of N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC 2.7.8.17). ^{1 2} This enzyme catalyzes the initial step in the synthesis of the mannose-6-phosphate (M6P) marker, which is essential for transferring hydrolases to lysosomes. ^{3 4} The deficiency of several lysosomal enzymes intracellularly and increased levels extracellularly (serum and plasma) have been observed, which results in the accumulation of their substrates. ⁴ However, there seems to be an alternative route to M6P, since some cell types have similar, close to normal, levels of lysosomal enzymes (leukocytes, liver, spleen, kidney, and brain). ⁵

The incidence of MLs II and III varies between different populations, from an estimated 1:120,000 of ML II in Portugal ⁶ to 1:252,000 in Japan. ⁷ As regard to MLs II and III, it ranges from 1:325,000 in Australia (including pre- and postnatal) ⁸ to 1:642,000 in the Netherlands. ⁹ Estimates of ML III α/beta prevalence are not available. In general, it was considered that the prevalence is in the same order of magnitude as that of ML II. ^{10 11}

Classification was based on clinical and molecular alterations. It is known that patients with ML II and patients with ML III α/beta have pathogenic mutations in GNPTAB , which encodes the α and β subunits of the enzyme, while ML III patients have mutations in GNPTG , which encodes the γ subunit of the enzyme, also known as ML III gamma. ¹²

The clinical manifestations of MLs II and III have not yet been fully elucidated, although they are essential in distinguishing type II from type III patients. They are known to be multisystemic, broad spectrum, and varied among affected individuals. Patients with ML II have presented earlier symptoms in their life, that are much more clinically evident at birth, and have a progressive course and a usually fatal outcome in childhood. ^{4 10 13} ML III α/beta patients show onset of symptoms in childhood, much slower progression and are usually fatal in early adulthood. Characteristic features are typical infiltrated face, gingival hyperplasia, flat nasal bridge, and severe developmental delay. In the skeletal system, they may present short, broad, claw hands, kyphoscoliosis, multiple dysostosis, and congenital hip dislocation. ^{4 10 13} Patients with ML III have a slowly progressive course, gradually coarse facial features, mild gingival hyperplasia; the language development is normal, and motor development may be normal or show minimal delay. The ML III gamma, however, is less described in the literature. The natural history of ML III is poorly described. ^{11 14 15}

Our understanding of MLs II and III is increasing from the literature, but it is still very limited. The objective of our study is to characterize the clinical manifestations of a representative cohort of Brazilian patients with MLs II and III.

Materials and Methods

This was a cross-sectional, multicenter study approved by Hospital de Clínicas de Porto Alegre (HCPA) ethics committee. The sample was of convenience and included 27 patients with MLs II and III biochemical and/or genetic diagnosis, confirmed in the Laboratory of Inborn Errors of Metabolism of the Medical Genetics Service of HCPA (LEIM-HCPA), between 1983 and 2013. LEIM-HCPA is the largest national reference center for diagnosis and management of LSD in Brazil, and probably concentrates the largest number of MLs II and III diagnoses in Latin America. Since direct measurement of GlcNAc-phosphotransferase enzyme activity is not available, the diagnosis of these patients was indirectly confirmed by the activity of lysosomal enzymes as α-L-iduronidase, β-galactosidase, β-glucosidase, α-mannosidase, β-glucuronidase, β-hexosaminidase, arylsulfatase B, and iduronate 2-sulfatase. Patients with high levels of enzyme in plasma and reduced in fibroblasts were included. Twenty-one patients had molecular diagnosis ( Table 1 ) and individuals without fibroblasts available for biochemical confirmation were included only if they had DNA analysis with pathogenic mutations in GNPTAB or GNPTG genes.

Table 1. Genotype of patients with mucolipidosis types II and III included in this study.

Patient	Gene	Genotype (cDNA)	Genotype (protein)	Phenotype
1 a	GNPTAB	c.[1123C > T];[?] b	p.[Arg375*];[?] b	ML II
2 a	GNPTAB	c.[1514G > A];[1759C > T]	p.[Cys505Tyr];[Arg587*]	ML III α/beta
3 a	GNPTAB	c.[1208T > C];[3503_3504delTC]	p.[Ile403Thr];[Leu1168QfsX5]	ML III α/beta
4 a	GNPTAB	c.[3503_3504delTC];[1154C > T]	p.[Leu1168Glnfs*5];[Ser385Leu]	ML II
5 a	GNPTAB	c.[242G > T];[2249insA]	p.[Trp81Leu];[Asn750Lysfs*8]	ML II
6 a	GNPTAB	c.[2269_2273delGAAAC] ;[2269_2273delGAAAC]	p.[Glu757Lysfs*2];[Glu757Lysfs*2]	ML II
7 a	GNPTAB	c.[2808A > G];[3503_3504delTC]	p.[ Tyr937_Met972del];[Leu1168Glnfs*5]	ML II
8 a	GNPTAB	c.[1196C > T];[3503_3504delTC]	p.[Ser399Phe];[Leu1168Glnfs*5]	ML III α/beta
9 a	GNPTAB	c.[3503_3504delTC];[?] b	p.[Leu1168Glnfs*5];[?] b	ML III α/beta
10 a	GNPTAB	c.[3503_3504delTC];[3503_3504delTC]	p.[Leu1168Glnfs*5];[Leu1168Glnfs*5]	ML II
11 a	GNPTAB	c.[3503_3504delTC];[3503_3504delTC]	p.[Leu1168Glnfs*5];[Leu1168Glnfs*5]	ML II
12 a c	GNPTAB	c.[3503_3504delTC];[3503_3504delTC]	p.[Leu1168Glnfs*5];[Leu1168Glnfs*5]	ML II
13	GNPTAB	c.[3503_3504delCT];[1208T > C]	p.[Leu1168Glnfs*5];[ p.Ile403Thr]	ML III α/beta
14 d	GNPTAB	c.[1208T > C];[1723G > A]	p.[Ile403Thr];[Gly575Ala]	ML III α/beta
15	GNPTAB	c.[3503_3504delTC];[3503_3504delTC]	p.[Leu1168Glnfs*5];[Leu1168Glnfs*5]	ML II
16	GNPTAB	c.[1931C > T];[3668_3670delCT]	p.[Thr664Ile];[Thr1223delT]	ML III α/beta
17	GNPTAB	c.[3503_3504delCT];[1514G > A]	p.[Leu1168Glnfs*5];[Cys505Tyr]	ML III α/beta
18	GNPTAB	c.[242G > T];[?] b	p.[Cys505Tyr];[?] b	ML III α/beta
19 e	GNPTG	c.[328G > T];[328G > T]	p.[Glu110*];[Glu110*]	ML III gamma
20 e	GNPTG	c.[328G > T];[328G > T]	p.[Glu110*];[Glu110*]	ML III gamma
21 e f	GNPTG	c.[244_247dupGAGT];[328G > T]	p.[Phe83*];[Glu110*]	ML III gamma

^a Patients 1 to 12 were described in Cury et al. ¹⁶

^bOne unidentified mutation.

^c This patient was studied in detail in Alegra et al. ¹⁷

^d This patient was studied in detail in Sperb-Ludwig et al. ²¹

^e Patients 19 to 21 were also described in Velho et al. ²⁷

^f This patient was studied in detail in Velho et al. ²⁸

Clinical and demographic data were preferably obtained by the same author ( n  = 16) or by the attending physician ( n  = 11) by completing a specific clinical record, reviewing the medical records and interviewing relatives. Low birth weight was defined as weight < 2.5 kg. At the beginning of the study (2011), four patients had already died, and another six died in the course of the study. Classification of ML type (II or III) was according to the judgment of the attending physician and was always reviewed by two medical specialists in MLs II and III. The classification of ML III in α/beta or gamma was performed according to the genotyping results of GNPTAB and GNPTG . ¹⁶

Results

The diagnosis of MLs II or III was confirmed in 27 patients (ML II n  = 15; ML III α/beta n  = 9; ML III gamma n  = 3–16 males and 11 females) through enzymatic analyses in fibroblasts or pathogenic mutations in GNPTAB or GNPTG . All patients were diagnosed after birth. Among them, 14 were born from 2000 to 2010 (5 being from 2000 to 2005 and 9 from 2006 to 2010). Considering that 32,996,065 births occurred in Brazil during that period (Brazilian Ministry of Health, Information System on Live Births—SINASC), the incidence of MLs II and III was at least estimated to be 1:2,357,000 live births in the period.

The 27 patients included in the study were recruited from 25 unrelated families, in which 2 had reported parental consanguinity (8%). All patients are Brazilian: nine were from the Southeast region, eight from the Northeast, five from the South, three from the Midwest, and two from the North. The median age at diagnosis was 2.7 years (interquartile range 1.8–7.3).

Clinical Manifestations

Mucolipidosis Type II

Fifteen patients had ML II. The results are presented in Table 2 and Fig. 1 . There was reported oligohydramnios in one gestation, without reports of other complications during prenatal care for pregnancies. However, information about cephalic perimeter and length at birth were available only for n  = 6 (32 ± 1.6 cm) and n  = 8 (47 ± 4 cm) patients, respectively. Initial symptoms were observed in early childhood ( n  = 7, thoracolumbar hump, thoracic deformity with narrowing and bulging of the thorax), joint restriction in upper or lower limbs ( n  = 5), weight deficit ( n  = 3), and developmental delay ( n  = 3).

Table 2. Main clinical characteristics of patients with MLs II and III included in the study.

	Type II	Type III α/beta	Type III gamma	Total	Comparison MLs II and III (α/beta; gamma)
N	15	9	3	27	–
Sex, M:F	6:9	8:1	2:1	16:11	–
Consanguinity ( n )	2	0	0	2	–
Age at diagnosis, y (mean ± SD)	2 ± 1	13.4 ± 13.5	29.2 ± 19	7.9 ± 11	p  = 0.007
Death age (mean ± SD)	6 (4 ± 1.9)	1 (20)	0	7 (6.6 ± 6.2)	Age: p  = 0.006
Type of parturition vaginal:cesarean (% cesarean)	3:7 (70%)	6:3 (33%)	2:1 (33%)	11:11 (50%)	p  = 0.004
Weight at birth, kg (mean ± SD)	2.6 ± 0.5 ( n  = 11)	3 ± 0.6 ( n  = 7)	3.8 ± 0 ( n  = 1)	2.8 ± 0.6 ( n  = 19)	NS
% patients with low weight (<2,500 g)	55% ( n  = 6)	13% ( n  = 1)	0	37% ( n  = 7)	–
Onset of symptoms, y (mean ± SD)	0.3 ± 0.3	7.4 ± 7	12.5 ± 7.5	3.2 ± 5.6 (median = 0.5)	p  = 0.006

Abbreviations: ML, mucolipidosis; NS, not significant; SD, standard deviation.

Fig. 1.

Phenotypic spectrum of patients with GlcNAc-phosphotransferase-deficient activity (MLs II and III). ( A–C ) ML type II (2-year-old patient), protruding abdomen, thoracolumbar hump, typical face, gingival hyperplasia, and joint contractures may be observed. ML, mucolipidosis.

All patients had craniosynostosis and none presented hydrocephalus or other indications for corrective surgery. Other symptoms were described in a smaller number of patients: dry hair ( n  = 2), hepatosplenomegaly ( n  = 2), abdominal diastasis ( n  = 1), bilateral inguinal hernia ( n  = 1), frequent respiratory infections ( n  = 1), myocardiopathy ( n  = 1), and oligohydramnios ( n  = 1). No corneal opacity or deafness was reported in any patient through clinical examination. Obstructive sleep apnea, diagnosed by polysomnography, was described in 5 of 14 patients.

All patients were delayed in developmental milestones, predominantly motor development. Six patients could walk without support and five spoke more than 20 words. Others were unknown. Twelve patients had passed away, in which eight were caused by respiratory complications.

Mucolipidosis Type III α/Beta

Nine patients had ML III α/beta. The results are presented in Table 2 and Fig. 2 .

Fig. 2.

( A–C ) ML type III α/beta (29-year-old patient), pectus carinatum, articular contracture in knees, shoulders, and discreet hands; infiltrated face, but with little intensity. ML, mucolipidosis.

There were no complications reported during prenatal care. Information on cephalic perimeter and length at birth were not available for all patients: at birth, the cephalic perimeter presented a mean of 34.5 ± 0.7 cm ( n  = 2) and the length of 49.6 ± 2 cm ( n  = 4). The initial symptoms were observed close to or in adolescence, being the main ones: limb articulation in the upper or lower limb ( n  = 5), spinal (lumbar or thoracic) pain ( n  = 4), and weight deficit ( n  = 3). Other skeletal alterations including increase of dorsal kyphosis, pectus carinatum and short neck ( n  = 6), speech delay ( n  = 1), and protruding abdomen ( n  = 1) were less frequent.

Delayed developmental milestones were observed in three patients, but—despite this difficulty in early life—all patients were able to attend regular school without too much difficulty, some of them even pursuing higher education. In our cohort, one patient passed away at 20 years of age because of pulmonary hypertension, restrictive chest alterations, and respiratory complications.

Mucolipidosis Type III Gamma

Three patients had ML III gamma. The results are presented in Table 2 and Fig. 3 . There were no reports of complications during the prenatal period of these patients. Unfortunately, no information was retrieved from birth regarding head circumference; the length was reported as 53 cm for one of them and unknown for others. The initial symptoms were observed in adolescence, being the main: joint limitation in upper or lower limbs ( n  = 3), pain or alterations in the spine ( n  = 1), and weight-stature deficit ( n  = 2). The three patients had alterations in the hip joint, two with bilateral hip prosthesis (at 35 and 37 years in one patient, and 40 and 41 years in the other).

Fig. 3.

( A–C ) ML type III gamma (41-year-old patient), joint contractures, elbows, shoulders, and hands; this patient has bilateral hip prosthesis. ML, mucolipidosis.

All subjects attended regular school and did not demonstrate apparent cognitive deficit. No deaths were reported, with the age of the patients being evaluated (21, 43, and 46 years).

Clinical Findings

Gingival hyperplasia was observed in all ML II patients, in two of eight ML III α/beta, and none of ML III gamma patients. Infiltrated face was also observed in all ML II, in four of eight ML III α/beta, and one of eight ML III gamma. In addition, all patients had short stature in proportion to age. As the height of both parents was not available in most cases, the target height was not calculated.

In the physical examination of the cardiovascular system, patients presented alterations in cardiac auscultation (systolic murmur), usually related to mild valvular insufficiency ( n  = 8/8), patent foramen ovale ( n  = 1/8, 10 months, ML II), mitral thickening ( n  = 4/8, one ML III), mild mitral regurgitation ( n  = 7/8), aortic valve stenosis ( n  = 2/8, both ML III, one with mild obstruction and aortosclerosis), and pulmonary valve insufficiency ( n  = 8/8). Left ventricular hypertrophy was seen in two patients with ML II: concentric hypertrophy in one (4 years) and a discrete increase in left ventricle in another (5 years). Pulmonary artery systolic pressure (PASP) > 30 mm Hg was seen in three of eight patients: two with borderline (ML III) and one with pulmonary hypertension (ML III, currently using continuous positive airway pressure [CPAP] and sildenafil, with good response), and the others with PASP < 30 mm Hg, one patient (ML II) used CPAP and needed tracheostomy.

The most prominent skeletal alterations were in the hip ( n  = 23/23), joint contracture in hands (variable intensity) in 18 of 22 patients, being that 3 of 22 were ML III α/beta and 1 ML III gamma. All had joint contractures in elbows and shoulders, with varying severity between types and between individuals of the same type. Alterations in the lumbar and thoracic spine were the most frequent. Bone alterations in an ML II patient, compatible with transient neonatal hyperparathyroidism are described in Alegra et al. ¹⁷

Ocular alterations were poorly studied, but formal ophthalmologic evaluation was available in 13 patients. Of these, the most relevant finding was pigment dispersion in the retina in an ML III gamma patient, not described in the literature. None of the individuals had opacification of the cornea or spots on the fundus of the eye.

Despite the protruding abdomen observed in all individuals, only one ML II patient presented hepatosplenomegaly upon physical examination. Umbilical hernia was reported in one patient (ML III gamma), and inguinal hernia was seen in 2 of 15 patients with ML II.

Detailed neurological examination was not described for most patients, but there was no report of seizures or spinal cord compression in the period. One patient with ML III gamma presented severe carpal tunnel syndrome, diagnosed clinically and by electromyography. Image of the encephalon was available for three patients (ML III α/beta = 1, ML III gamma = 2).

In the respiratory system, which was the main cause of morbidity and mortality of individuals, chest restrictions were observed in all individuals, except for an ML III gamma. The number of hospitalizations or respiratory infections per year was not obtained, but by the subjective report of the patients, it appeared to be similar to the population, but when they occurred, they generally caused hospital admission. Obstructive sleep apnea was described in 5 of 14 patients; however, polysomnography to study this alteration was not always available to patients, usually due to difficulty related to the structure of the public health system.

When comparing ML III α/beta and gamma, we observed that the onset of symptoms was significantly earlier in ML III α/beta than in gamma (4.1 ± 4 vs. 7.6 ± 4.4 years, p  = 0.045), but there was no statistically significant difference as to the age at diagnosis (10.3 ± 7.6 vs. 29.3 ± 19.1 years, p  = 0.2).

Discussion

This is the most comprehensive clinical study exploring characteristics of a Brazilian cohort with MLs II and III, including the analyses of 27 patients. The only clinical study of MLs II and III published in Latin America reports five patients from São Paulo. ¹⁸

ML II predominated in our sample in relation to ML III. In the literature, it is not clear which type of ML is more frequent, ^{4 13} but some authors suggest that the frequency of both is similar ^{10 11} ; as ML II is clinically more severe, this could increase the search for diagnosis and, consequently, the number of individuals diagnosed. The diagnosis is really challenging. ¹⁹ We described the difficulties found in the prenatal diagnosis of a heterozygous individual, the brother of a patient in our cohort. ²⁰ In one of the patients, with very mild phenotype suggesting ML III, the diagnosis was just possible using whole genome sequencing. ²¹ The overall inbreeding rate in our sample was higher than that observed in the literature (8 vs. 1.5–0%). The incidence may vary when inbreeding occurs, as in Ireland, where the prevalence of ML is 1.56:100,000 live births and in “Irish Travelers,” a population with a high rate of inbreeding is 114:100,000. ^{22 23}

There was a high cesarean rate in both groups (50% in total), which is similar to the Brazilian rate of 43.8% of cesarean childbirth (source: Ministry of Health, SVS—Information System on Live Births/2009). In the comparison between the subgroups ML II and ML III, there were more cesareans in the ML II ( p  = 0.006), whose ratios we do not know, since data are not available. There were no statistically significant differences between the two groups at birth, but patients with low weight (<2,500 g) were predominant in ML II patients (55%, n  = 6) in contrast to ML III α/beta patients (13%, n  = 1) and the Brazilian population (8.43%, source: Ministry of Health, SVS—Information System on Live Births/2009). Cathey et al ¹³ observed mean birth weight of 2,400 g for ML II and 3,100 g for ML III (α/beta), being 8 of 12 ML II patients small for gestational age.

As our data were retrospective, there was loss of information from the pre- and perinatal periods, including birth conditions, for example. Through our analysis, we observed that most ML II patients presented weight <2,500 g at birth (none weighing <1,700 g), but unfortunately, we did not have information on gestational age to correlate to weight and to understand if the patients were also small for the gestational age or whether these data correlated with prematurity.

The age of the first symptom in our patients was different from the literature, being in our case, the presentation noticed later, and in the largest available clinical study, all ML II patients and 5 of 21 ML III α/beta patients had symptoms at birth. ¹³ This difference could be attributed to the fact that it is a rare disease with low incidence in Brazil, so most clinicians are not aware of its existence and mild features could go unnoticed in the first day of life. Thus, it is important for clinicians to be aware that infants with joint restriction and coarse facial features might need further investigation regarding rare diseases, including mucolipidosis.

The prominent symptom in all individuals was joint restraint, which we could observe objectively through goniometry in a small number of patients; the most affected joints of this sample were knees (flexion), hip, and ankle, with worse performance of ML III patients, probably because they were older than ML II patients. When comparing ML III α/beta and gamma, we observed that the age of onset of symptoms was significantly lower in the ML III α/beta and with more severe somatic involvement, which was not observed by Nampoothiri et al. ²⁴

Cognition has not been measured with formal instruments, but ML II has motor development and language quite affected concordant with the literature. ^{13 23 25} ML III α/beta and gamma have already achieved satisfactory schooling for their age, although the former showed a delay in motor development in early life. As we do not measure these data in a formal way (with the intelligent quotient [IQ]), we cannot compare directly in the literature. It is described for ML III α/beta cognitive deficit in 5 of 15 patients and for ML III gamma in 1 of 8 (IQ lower than 70). ^{13 26}

This is the most comprehensive clinical study exploring characteristics of Brazilian patients with MLs II and III. ML II was more frequent and with more severe and precocious somatic and cognitive impairment. ML III α/beta patients present greater somatic impairment than ML III gamma and developmental delay only in the first year of life. Some practical issues of care are still controversial among international authors and require further answers. In addition, we believe that a better understanding of the natural history of the disease may contribute to the ongoing therapies and future treatments that may arise.

Funding Statement

Funding Financial support for this project was provided by FIPE-HCPA, FAPERGS, CAPES, CNPq (Brazil), and the UFRGS Graduate Program in Genetics and Molecular Biology.