The age range of cases was 2 days to 56 years and age of onset of symptoms was from in-utero to 15 years. Affected individuals were 21 males and 10 females making the male to female ratio 2:1 (65.51% males and 34.48% females). Three couples assessed were not included in the ratio calculation.
Pedigree analysis indicated that of the 34 cases, 15 (44.11%) had positive family history with a first/second degree relative who had same or similar disorder, while 19 cases (55.88%) were sporadic. From the pedigree, we estimated five cases to be AD, of which four were confirmed, while the fifth one was an XD disorder confirmed after NGS testing. Fifteen cases were predicted to have AR inheritance, of which 14 were confirmed by the molecular report, whereas the last case was identified to have an XR condition in a child. Only one pedigree showed an XR which was confirmed after molecular testing. Consanguinity was documented in 15 (44.11%) families suggesting AR inheritance, however, only 12 out of 15 consanguineous cases had a confirmed AR disorder post molecular diagnosis (Tables 1 and 2). AD cases identified were 15, XR were 3 and one family was diagnosed to have XD disorder.
Clinical spectrum of the cases
The cases which were evaluated by NGS were broadly classified into disorders involving musculoskeletal system and storage disorders depending on whether or not they showed systemic involvement.
Disorders involving Musculoskeletal system – 76% (26 of the 34) cases were identified as . musculoskeletal disorders with genetic basis after radiological and clinical criteria and were categorized as:
a. Osteogenesis Imperfect (OI)
Osteogenesis imperfecta (OI) is a genetic disorder affecting the connective tissue and is mainly characterized by extremely fragile/brittle bones that fracture with trivial traumas 9. OI is of more than ten types based on clinical features that often overlap. Severity varies with type I being the mildest form and type II being the severest form. COL1A1 and COL1A2 gene mutations are responsible for more than 90% of all cases of OI, however, other genes have also been associated 10.
The eight cases (1.1.1-1.1.8 cases) clinically diagnosed as OI had common features such as recurrent fractures and blue sclera. Their age of disease onset was from in-utero to 15 years. Seven of the patients were born of non-consanguineous marriage and four of these had another family member affected (Table 1.1). Four out of eight cases were identified (50% diagnostic yield) with pathogenic heterozygous sequence variant in COL1A1 or COL1A2 genes indicating AD inheritance. A homozygous intronic variant in COL1A2 gene which is known to be polymorphic and may be associated with phenotype was found in a 5-year-old, with childhood onset recurrent fractures, blue sclera, and a positive family history. Two cases were identified with homozygous Variant of uncertain significance (VUS) in COL1A1 gene and SERPINF1 gene, it is interesting to note that the case 1.1.5 with COL1A1 who had homozygous VUS also exhibited additional clinical features like dentogenesis imperfecta, slurred speech, dysmorphism and mild scoliosis indicating the severity likely caused due to homozygosity. Segregation analysis may be useful to understand the significance of these variants. However, no significant variant correlating with phenotype was identified in case 1.1.8.
Osteopetrosis is a rare genetic disorder characterized by an increase of bone mass due to defective osteoclast function. Patients typically display spontaneous fractures, anemia, and in the most severe forms hepatosplenomegaly and compression of cranial facial nerves leading to deafness and blindness 11. We confirmed diagnosis of two cases of osteopetrosis (1.2.1-1.2.2). A heterozygous pathogenic variant in CLCN7 gene indicating Osteopetrosis type 2 (1.2.1) and the other, a VUS in LRP gene known to cause Osteopetrosis type 1 (case 1.2.2)
c. Split-hand/foot malformation-6
Split Hand/Split Foot Malformation or Ectrodactyly is a genetic disorder characterized by the complete or partial absence of some fingers or toes, often combined with clefts in the hands or feet 12. Two sisters (1.3 case) with ectrodactyly born to consanguineous parents were identified with homozygous pathogenic variant in WNT10B gene confirming the diagnosis of split hand/foot malformation 6.
d. Skeletal dysplasia with in-utero presentation
It is well established that DMS can be identified in the prenatal period. Two POC, one perinatal death and three couple samples were assessed in this category. Couple samples were analysed because of BOH with skeletal involvement and unavailability of samples from the affected fetus. Analysis of the male fetal sample (case 1.4.1) revealed hemizygous VUS in MTM1 gene diagnostic of XR myotubular myopathy, which is associated with skeletal dysplasia and polyhydramnios in utero, both characteristics, were reported in the ultrasound report available 13. Since the couple was planning their next child, the mother was evaluated and was identified to have the same variant in heterozygous state confirming carrier status. This also explained the childhood death of two maternal uncles which was documented in the pedigree. Hence this MTM1c.413C>T p.Thr138Met variant can be considered as a novel pathogenic variant after confirmation with functional analysis.
The second POC (case 1.4.2) revealed a novel pathogenic homozygous variant c.867_877del causing protein truncation p.Gln289HisfsTer2 in CUL7 gene causative of 3M syndrome, which has a phenotype of severe prenatal and postnatal growth retardation, long, slender tubular bones, reduced antero posterior diameter of the vertebral bodies and delayed bone age 14. While the ample from a neonate who passed away (case 1.4.3) with a clinical diagnosis of Dandy walker with radiological evidence of malformation and oligohydramnios exhibited two compound heterozygous VUS (which included novel variant c.280+7_280+9AG) in POMT1 gene causative of Walker-Wurburg syndrome 15 .Segregation analysis was recommended in both cases
Three couples were screened by NGS in view of consanguinity and BOH with DMS. Two couples were identified to be carriers for AR disorders with single heterozygous causative variant whereas one couple had different causative variants in same gene contributing to compound heterozygosity in offspring. Case 1.4.4 was identified with compound heterozygosity comprising of a novel pathogenic (c.6134C>T, p.Ser2045Leu) variant and VUS in CEP290 associated with Joubert syndrome 5 (OMIM # 610 188). Case 1.4.5 revealed heterozygosity in both partners for likely pathogenic variant in RBBP8 causative of Seckel Syndrome 2 (OMIM # 606744) whereas case 1.4.6 revealed both parents to be heterozygous carriers for a VUS in COL11A2 associated with Fibrochondrogenesis 2 (OMIM # # 614524).
e. Short stature
Although short stature is observed in most skeletal dysplasia cases, four of our syndromic cases were found to have short stature in addition to other features. A female member of a family where multiple members had renal rickets was tested and identified to have a pathogenic variant in PHEX gene confirming the diagnosis of X linked dominant Hypophosphatemic rickets 16.
Other three pediatric cases had short stature as a part of a developmental disorders, a homozygous pathogenic variation in ERC66 gene associated with Cockayne syndrome B 17,cerebro oculofacio skeletal syndrome 1 was identified in case 1.5.2, while the third case exhibited VUS in CUL4B gene causative of Cabezas type of X-linked syndromic mental retardation 18 and the fourth case was identified to have heterozygous VUS in AHDC1 associated with Xia Gibbs Syndrome 19.
f. Joint stiffness and pain
Two cases of arthritis as a part of systemic disorders were evaluated in this study. Case 1.6.1 of 21 year old male with clubbing of digits, Erlenmeyer flask deformity of distal tibia and fibula, thrombocytopenia and bilateral polycystic kidneys was identified to have a novel homozygous pathogenic variant c.325delG causing protein truncation p.Ala109LeufsTer64 in SLCO2A1 gene diagnosing it as primary hypertrophic osteoarthropathy, autosomal recessive 2 (PHOAR2) 20 .It is interesting to note that the same individual was also identified with a heterozygous pathogenic variant in PKD2 gene c.637C>T p.Arg213Ter associated with AD polycystic kidney disease explaining the renal phenotype. Case 1.6.2 was identified with a pathogenic homozygous variant in CCN6 gene causative of autosomal recessive pseudorheumatoid dysplasia manifesting as joint stiffness since the young age of 16 years 21.
A female who had joint restrictions and genu valgum and avascular necrosis was identified to have a pathogenic variant in COL9A1 gene associated with multiple epiphyseal dysplasia 22. A 8 months old male born to non-consanguineous parents presented with hyperpigmentation and joint swellings and was reported to have novel likely pathogenic duplication c.54_60dupGCTGTGG causing premature truncation (p.Leu21Alafs*26) in ANTXR2 genecausative of hyaline fibromatosis, a condition where there is deposition of amorphous hyaline material in skin and visceral organs 23. A 30 year old female with family history of cancer who has café au lait spots was assessed and found to have a variant in NF1 gene associated with neurofibromatosis 24.
2. Storage disorders
Lysosomal storage disorders (LSDs) are a large group of more than 50 different inherited metabolic diseases which, in the great majority of cases, result from the defective function of specific lysosomal enzymes and non-enzymatic lysosomal proteins or non-lysosomal proteins involved in lysosomal biogenesis 25.
a. Mucopolysaccharidoses (MPS)
MPS is characterized to seven subtypes and are caused by deficiencies in the lysosomal enzymes necessary for the degradation of glycosaminoglycans (GAGs). Storage of GAGs affects the skeletal tissue, cartilage and connective tissues, as well as the peripheral and central nervous system 26.
Of the five cases clinically suspected with MPS, three were consanguineous. Pathogenic variations in IDUA were identified in two cases diagnosing MPS I. A novel hemizygous likely pathogenic missense variation c.1493 G>C causing protein change p.Arg498Thr was identified in IDS gene in a 2yr/male patient confirming MPS II. Two brothers with knock knees and h epatosplenomegaly had a likely pathogenic homozygous variant in GALNS gene which is associated with MPS IVA. Another 7-year-old male with intellectual disability, corneal clouding was identified to have a homozygous VUS in GUSB gene causative of MPS VII. The younger brother of the proband was identified with the same variant in GUSB gene followed by enzyme analysis showing deficient β-Glucoronidase activity thereby confirming the pathogenicity of the VUS identified.
Mucolipidosis has the clinical and biochemical features of both MPSs and sphingolipidoses, being characterized by the accumulation of glycoproteins and glycolipids 26. In our cohort, there were two clinically cases suspected of mucolipidosis with consanguinity in one case. Pathogenic GNPTG homozygous variant was identified in a 7yr/female. Two VUSs in GNPTAB with compound heterozygosity were identified in another female child of 4 years. A 9-year-old female (2.2.3) born to non-consanguineous couple with storage disorder like symptoms and elevated urine GAGs was suspected to have mucolipidosis in view of no likely causative variants in MPS genes, however, no diagnostic variant was identified to confirm mucolipidosis in this patient.