Genetic epidemiology, mapping and molecular characterization of families with inherited skeletal and neurological disorders

Abstract

Congenital and hereditary anomalies (CA) are common in Pakistan and put a tremendous impact on the morbidity and mortality of infant, young and adult populations. The present study aimed to investigate the epidemiological, clincal and molecular genetic characteristics of Pakistani families affected with CA. This multitiered study was carried out in order to: 1) estimate the burden of CA in the Hazara population of Pakistan by determining their prevalence-pattern and bio demographic coorelates; 2) establish the prevalence-pattern, phenotypic manifestation and descriptive genetics of congenital limb deficiencies; 3) clinical and molecular diagnosis of rare malformations segregating in extended families; and 4) provide clinical and functional evidence through animal model that rare variant is causative for craniofacial anomalies. First, through a mix of modified random door-to-door and convenience sampling approach, 1,189 independent cases/families with CA were identified in the Hazara population (Chapter 2). The estimated consanguinity rate in this sample was 66%. An estimated 65% cases were sporadic in nature and 35% familial; and 70% were isolated and 30% syndromic. CA were categorized into 9 major phenotypic types. Neurological disorders were the most common (n=486; proportion=0.409;% CI=0.381-0.437), followed by limb defects (n=292), musculoskeletal defects (n=106), sensorineural/ear defects (n=101), blood disorders (n=75), eye/visual impairments (n=39), ectodermal anomalies (n=30), and congenital heart defects (n=26). This is the first study of its kind reporting prevalence-pattern of CA in Hazara population of Pakistan. Congenital limb deficiencies (CLD) are very rare anomalies and cause severe disability. In another study (Chapter 3), 141 independent subjects involving 166 limbs with CLD were recruited from Khyber Pakhtunkhwa (KP) province of Pakistan. Subjects with transverse defects were n=77 (55%), longitudinal defects n=61 (43%), and intercalary defects n=3 (2%). Upper limb deficiencies were 86% compared to lower limb deficiencies, i.e., 14%. There was a high preponderance of unilateral cases (83%), sporadic nature (92%), low parental consanguinity rate (33%), and syndromic appearance in 21% of cases, which may be indicative of the substantial role of non-genetic factors in the etiology of CLD. This is the first study reporting phenotypic pattern of CLD from Pakistan. Increased inbreeding is a common cause of the recessive mutations that lead to the appearance of rare disorders in Pakistani families. Another aim of the current research xv was to locate the causal mutations in Pakistani families associated with autosomal recessive malformations. Here, four families with rare hereditary skeletal, neurological and ophthalmological disorders were recruited. DNA of the available affected and unaffected subjects was extracted from peripheral blood for molecular study. Whole genome SNP genotyping and/or exome sequencing were carried to in order to find the candidate chromosomal region/variant. (i) In the first family (Chapter 4), the patients were presented with intellectual disability, developmental delay, orofacial defects and a range of behavioral anomalies. In this family, SNP based homozygosity mapping led to the discovery of 12 homozygous intervals >1Mb, the largest (54Mb) on chromosome 5p15.2-q12.3, followed by 39Mb stretch on 3q23 q26.32 and 22Mb on 4q31.22-q32.3. Several potential candidate genes related to the phenotype were present on these regions. These findings would be very helpful in a prospective gene hunt through exome sequencing. (ii) In the second family (Chapter 5), a 13-year old boy was presented with cryptophthalmia, midface hypoplasia, agenesis of right kidney, and cutaneous syndactyly in fingers and toes but no symptoms in any other organ including lungs, anorectal system, genitalia or umbilical system were observed. Exome sequencing in this family led to the discovery of homozygous truncating variant c.1774C>T (p.Gln592Ter) in GRIP1 gene which is known to cause Fraser syndrome 3 (FRASRS3). This case is also the oldest reported individual with FRASRS3, to our knowledge, and shows that FRASRS3 case may be milder than known and live into at least adolescence. (iii) In family 3 (Chapter 6), the patients were presented with rhizomelia, short trunk dwarfism, scoliosis, microcephaly, intellectual disability, and other clinically diverse symptoms. Exome sequencing in this family led to the discovery of a novel homozygous missense mutation c.1072C>T (p.Q358*) in DYMECLIN gene. This mutation was predicted to form a truncated DYMECLIN (DYM) protein which may profoundly impair its normal function. The mutation in this gene is known to cause Dyggve-Melchior Clausen disease (DMC). The current molecular analysis described a putative DMC phenotype associated with a DYM gene and also confirmed the clinical condition that segregate in the family. This study findings provide a start point for future studies on the function of DYMECLIN protein. (iv) In family 4 (Chapter 7), three affected subjects were presented with short neck, disproportionately short trunk, and protruding abdomen (secondary to lordosis). Other common characteristics included kyphoscoliosis, winged scapulae, crowded ribs, and pectus carinatum, while xvi clinodactyly was present in one of the affected individuals. Exome analysis revealed homozygous missense mutation c.220C>T (p.Glu74*) in the coding region of FLNB gene segregating with the malformation. Conclusively, the study described the expanded clinical spectrum of spondylocarpotarsal synostosis (SCT) syndrome. The scientific findings of this study might be helpful to establish genotype-phenotype correlation of SCT. Lastly (Chapter 8), a family with a single female patient was presented with syndromic condition along craniofacial anomalies like, cupid’s bow, small jaw, and narrow palate. Exome sequencing in this subject led to the discovery of de novo compound heterozygous mutations p.L605P and p.M319V in the CFAP46 gene. In order to get an insight into the molecular function of this gene, the disease was modeled in vivo using CRISPR/Cas9-mediated genome editing in frog tadpoles, and the functional effects of the detected variant on candidate protein function were evaluated. The whole mount in situ hybridization technique was adapted to explore the role of the CFAP46 protein in embryonic development. The craniofacial anomalies induced by the CRISPR/Cas9-mediated genome editing of CFAP46 mimicked the patient's condition. Since the non-overlapping CRISPRs were so effective, it is likely that CFAP46 is crucial to development. Further evidence from whole-mount in situ hybridization indicated that CFAP46 is essential for neural crest development and that abnormalities of the face can result from CFAP46 protein deficiency. This work presents the first evidence of human craniofacial anomalies caused by CFAP46 mutations and provides clinical and functional support for this hypothesis. In conclusion, the findings of this study may be helpful for understanding the puzzling nature of the rare genetic disorders. This knowledge could result in cutting-edge methods for quickly diagnosing, treating or preventing rare diseases. Clinicians might benefit from the study, and it would be equally useful for other medical institutes and organizations as well as for setting up centers for genetic counseling.