Novel splice mutation in LRP4 causes severe type of Cenani-Lenz syndactyly syndrome with oro-facial and skeletal symptoms

Novel splice mutation in LRP4 causes severe type of Cenani-Lenz syndactyly syndrome with oro-facial and skeletal symptoms

Accepted Manuscript Novel splice mutation in LRP4 causes severe type of Cenani-Lenz syndactyly syndrome with oro-facial and skeletal symptoms Muhammad...

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Accepted Manuscript Novel splice mutation in LRP4 causes severe type of Cenani-Lenz syndactyly syndrome with oro-facial and skeletal symptoms Muhammad Afzal, Qamar Zaman, Uwe Kornak, Stefan Mundlos, Sajid Malik, Ricarda Flöttmann PII:

S1769-7212(17)30004-6

DOI:

10.1016/j.ejmg.2017.05.004

Reference:

EJMG 3293

To appear in:

European Journal of Medical Genetics

Received Date: 11 January 2017 Revised Date:

24 May 2017

Accepted Date: 25 May 2017

Please cite this article as: M. Afzal, Q. Zaman, U. Kornak, S. Mundlos, S. Malik, R. Flöttmann, Novel splice mutation in LRP4 causes severe type of Cenani-Lenz syndactyly syndrome with oro-facial and skeletal symptoms, European Journal of Medical Genetics (2017), doi: 10.1016/j.ejmg.2017.05.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Afzal et al.

Novel Splice Mutation in LRP4 causes severe type of Cenani-

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Lenz Syndactyly Syndrome with Oro-facial and Skeletal Symptoms

Muhammad Afzal,1 Qamar Zaman,1 Uwe Kornak2,3,4, Stefan Mundlos 2,3,4, Sajid

Human Genetics Program, Department of Animal Sciences, Faculty of Biological Sciences,

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Malik,1* Ricarda Flöttmann2**

Quaid-i-Azam University, Islamabad, Pakistan 2

Institut fuer Medizinische Genetik und Humangenetik, Charité-Universitaetsmedizin Berlin,

Berlin, Germany.

Max-Planck-Institut fuer Molekulare Genetik, FG Development & Disease, Berlin, Germany.

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Berlin-Brandenburg Center for Regenerative Therapies, Charité-Universitaetsmedizin Berlin,

Berlin, Germany.

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*Correspondence to: Sajid Malik, Human Genetics Program, Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan. Tel: +92-51-

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90643158. E-mail: [email protected]

**Correspondence to: Ricarda Flöttmann, Institut für Medizinische Genetik und Humangenetik, Charité, Berlin, Germany. Tel: +49-30-450-569121. E-mail: [email protected]

Running title: Novel LRP4 mutation causes Cenani-Lenz syndactyly

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The authors declare no conflicts of interest.

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Conflict of interest

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ABSTRACT

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Cenani-Lenz syndactyly syndrome (CLSS; MIM-212780) is a rare autosomal recessive limb malformation characterized by complete osseous fusion of all fingers and toes, disorganization of phalangeal elements and severe shortening of the radius and ulna. It is occasionally associated with renal hypoplasia, oro-facial defects, scoliosis of the thoracic spine, hearing loss, and genital

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anomalies. Here we describe a consanguineous Pakistani kindred with a severe form of CLSS

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characterized by complete syndactyly and disorganization of fingers, oligo-syndactyly of toes, shortening of limbs, frontal bossing, and hypoplasia/agenesis of left kidney. The affected individuals were additionally presented with short stature, cleft-lip and hypoplastic shoulder joint with restricted upper limb movement. A novel splice variant in LRP4 (c.316+1G>A) segregated with the phenotype in a five generations family. The mutation is predicted to add 29 non-native

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amino acids with a premature termination, resulting in approximately 90% length reduction of the wild-type transcript. These findings not only further expand the phenotypic variability of CLSS but also indicate that early truncated and loss-of-function mutations in LRP4 lead to a

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more severe CLSS phenotype.

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Key words: Cenani-Lenz syndactyly; LRP4; short stature, shoulder joint; cleft-lip; Pakistani family

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INTRODUCTION Cenani-Lenz syndactyly syndrome (CLSS; MIM-212780) is a rare autosomal recessive limb

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malformation initially described by Turkish and German colleagues A. Cenani and W. Lenz (1967). It is characterized by complete osseous fusion of all fingers and toes, shortening of the radius and ulna with fusion, and disorganization of phalangeal development (Temtamy and

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McKusick, 1978). The feet are less severely affected. The phenotype is usually bilateral and symmetrical. Of all the syndactylies known it is one of the most severe types (Harpf et al. 2005;

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Malik, 2012).

Seven et al. (2000) reported a patient who, in addition to the typical CLSS symptoms, also exhibited scoliosis of the thoracic spine and mixed hearing loss. Temtamy et al. (2003) reported two probands with CLSS and mild facial dysmorphism. In one of the families a

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similarly affected sibling with genital anomalies and cleft palate was reported. Jarbhou et al. (2008) described a patient with CLSS with facial dysmorphism, hypoplasia of the kidney, hyperthyroidism, laryngomalacia, and congenital dislocation of hips; thus further expanding the

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phenotypic manifestation of CLSS.

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Homozygous or compound heterozygous mutations in LRP4 at chromosome 11p11.2 have been implicated in the majority of cases with CLSS (Li et al. 2010). In 3 unrelated consanguineous Turkish families, CLSS segregated with a homozygous 1585G-A transition in exon 13 of LRP4. In 2 unrelated consanguineous Egyptian families, a 409G-A transition in exon 4 was detected. Other cases with the recessive type of CLSS were observed with homozygosity for 547+1G-A transition in intron 6, a 479G-A in exon 5, 1345G-A transition in exon 12, and 1382A-C transversion in exon 12 of the LRP4 (Li et al. 2010). CLSS phenotypes with dominant

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and recessive inheritance patterns have been shown to be caused by genomic rearrangements of GREM1-FMN1 locus and mutation in APC, respectively (Dimitrov et al. 2010; Patel et al. 2015).

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Herein, we report a novel splice-site mutation c.316+1G>A in intron 3 of LRP4 in a

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family of Pakistani origin with a severe CLSS phenotype with oro-facial and skeletal anomalies.

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SUBJECTS The family originates from a rural area of central Punjab, Pakistan. A pedigree of the

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family was constructed with the help of elders of the family. Three affected and 5 unaffected subjects were examined with the help of local physicians. Photographs of three and X-ray films of two subjects were obtained. Peripheral blood samples from three affected and four unaffected subjects were available for molecular study. All the material was collected after informed

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consent according to the Helsinki II declaration. The study was approved by the ethical review

MOLECULAR METHODS

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committee of Quaid-i-Azam University.

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LRP4 gene was analysed by Sanger sequencing using standard methodology.

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CLINICAL REPORT The pedigree spanned five generations and three of five siblings exhibiting CLSS

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segregated in the fifth generation (Fig. 1A). Parents of the affected individuals were consanguineous.

The affected subjects had a severe CLSS phenotype, associated with oro-facial findings,

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hypoplastic shoulder joint and mildly short stature. The limb phenotype was characterized by bilateral total fusion of fingers resulting in shortening of zeugopods and stylopods (Fig. 2B,G,H).

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The radiographs revealed short humeri with hypoplastic heads, profound shortening and synostosis of radii-ulnae, and hypoplasia/aplasia of carpals, metacarpals and phalanges. None of the osseous elements in the hands were recognizable. There was limited extension and flexion movement of the elbows. Shoulder joints were hypoplastic. The radiographs revealed delayed

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bone age in the shoulder joint, dysplastic proximal heads of humerii, malformed scapulae and malpositioned clavicle (Fig. 2J). The affected subjects were unable to lift heavy objects. Mild thoracic scoliosis was also evident (Fig. 2J).

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The feet were oligodactylous and reduced in size, bilaterally (Fig. 2C,E,K). Preaxial

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cutaneous fusion of toes was also evident (Fig. 2E). Radiographs revealed three phalangeal rays in the feet and the first digital rays appeared hypertrophic. Subject 505 had difficulty in walking. The radiographs of her legs showed hypoplasia of knee joints and posteriorly displaced fibulae (Fig. 2F,K). The abnormality in the lower limbs resulted in mildly short stature (compared with the growth standards of Pakistani subjects and the unaffected family members; Table 1). The affected subjects exhibited oro-facial symptoms like broad forehead, high hair-line, deformed pinna, and antevert/short nose with depressed nasal bridge (Fig. 2A,D,G). Frontal 8

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bossing with increased OFC was evident (Table 2). One of the subjects (502) with cleft-lip had undergone surgical correction and was presented with mal-aligned frontal teeth (Fig. 2A). A minor depression on the nasal tip of one of the subject (504) was notable (Fig. 2D).

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Ultrasonographic examination revealed hypoplasia of left kidney in subject 502 and agenesis of left kidneys in 504 and 505. The clinical features and anthropometric measurements of the

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family members are summarized in Tables 1 and 2.

Reportedly, another subject in the family (302) had cleft-lip without any limb phenotype

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(Fig. 1A); however, he was not available for clinical examination. The affected subjects had normal vision, hearing and speech. They were attending formal schools and adopted to perform

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routine life tasks.

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RESULTS Initially, the index was subjected to Sanger Sequencing of LRP4. A splice variant

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NM_002334: c.316+1G>A in intron 3 (Chr11: 46921812; UCSC-hg19) was detected (ClinVar: SUB2507338). This variant was observed in a homozygous state in the index and the two affected siblings while all other probands carried the variant in a heterozygous state (Fig. 1B).

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The retained intron 3 would add 29 non-native amino acids in the protein with a premature stop codon leading to a truncated protein. The detected novel variant is bioinformatically predicted to

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be disease-causing (online tools MutationTester, NetGene2, NNSPLICE), and is found in the general population with a very low heterozygote frequency of approximately 1/20,000 (ExAC database). Therefore, the variant is considered to be causative for the observed phenotype (Supp.

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Table 1).

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DISCUSSION LRP4 encodes Low-Density Lipoprotein Receptor-Related Protein 4. It mediates SOST-

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dependent inhibition of bone formation and inhibition of Wnt signaling (Xiong et al. 2015). LRP4 has been shown to antagonize LRP5/LRP6 activation of WNT/β-catenin signaling (Li et al., 2010; Leupin et al. 2011). LRP4 functions as a cell surface endocytic receptor binding and internalizing extracellular ligands for degradation by lysosomes (Dietrich et al., 2010). In the

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developing limb, it is involved in the dorsal/ventral pattern formation (BioGraph). Loss of LRP4

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function leads to excessive Wnt and β-catenin signaling in the limb bud, leading to abnormal development. Disruptions in the Wnt/β-catenin pathway result in a wide range of pathologies and congenital malformations including cancers (Johnson et al., 2006; Clevers and Nusse, 2012). In addition to CLSS, compound heterozygous missense mutations in LRP4 cause congenital myasthenic syndrome 17 (MIM-616304) (Ohkawara et al. 2014), and homozygous or

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heterozygous mutations cause sclerosteosis 2 (MIM-614305) (Leupin et al. 2011).

Most of the reported mutations in LRP4 are missense (transitions/transversions) or splice

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site mutations and do not result in immediate frameshift and/or premature truncation of the RNA transcript (Li et al. 2010). These mutations were shown to be associated with rather mild CLSS

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phenotypes with minimal involvement of the ulnae and radii. We report on a family with a severe CLSS phenotype. The novel splice variant in LRP4

(c.316+1G>A) segregates with the phenotype. The missense variant adds 29 non-native amino acids with a pre-mature stop-codon. This would result in approximately 90% length reduction of the wild-type transcript. The early truncated protein would consist of only the LDL repeats and would lack the various essential binding domains in LRP4. Further, it remains unknown whether

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the truncated RNAs would lead to short versions of proteins with residual function or if the transcripts undergo nonsense mediated decay. We did not perform any functional assays to further characterize the mutation. We used three different bioninformatical prediction tools:

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MutationTaster, NetGene2 and NNSPLICE. All three tools predicted the truncated variant to be disease causing due to splice site changes. None of those tools predicted nonsense-mediated RNA decay and there was an even shorter protein reported in the literature (Kariminejad et

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al.,2014). The truncated proteins are predicted to be abolished or pathogenic, while in the case of non-sense mediated decay, it would result in a complete lack of functional protein. Functional

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studies are required to elucidate the consequences of these mutations.

The patient reported by Kariminejad et al. (2013) also exhibited a phenotype on the severe end of CLSS spectrum, i.e., hypoplastic and synostosed radii/ulnae, oligodactyly in hands

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and feet, hypoplastic tibiae/fibulae, hearing loss, renal hypoplasia, high arched palate, enamel hypoplasia and supernumerary nipples. In the present family, hearing loss, high arched palate, enamel hypoplasia and supernumerary nipples were not witnessed. On the other hand, short

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stature, scoliosis, cleft-lip and hypoplasia of shoulder joint were witnessed in our family. Recently, compound heterozygous truncating mutations in LRP4 have been shown to be

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associated with a prenatal lethal phenotype of CLSS (Lindy et al. 2014). In two sibling fetuses Linddy et al. (2014) observed mesomelic limb reductions, oligosyndactyly, genitourinary malformation, rib anomalies and renal hypoplasia/aplasia. Taken together, the data of these three independent families suggest that early truncation of LRP4 lead to severe CLSS manifestations. It is not known whether the truncated RNAs would lead to short versions of proteins with residual function or if the transcripts undergo nonsense mediated decay. In all the three cases, the

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truncated proteins are predicted to be abolished or pathogenic, while in the case of non-sense mediated decay, it would result in a complete lack of functional protein. Functional studies are required to elucidate the consequences of these mutations.

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In conclusion, the present case adds evidence to the notion that truncating LRP4 mutations are associated with more severe CLSS phenotype involving mesomelic limb

shortening and malformation in several other organ-system, compared with the missense and

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splice site mutations that do not lead to early truncations and have less damaging effects. These

ACKNOWLEDGMENTS

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findings, therefore, support the genotype–phenotype correlations raised in previous reports.

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We thank the family members for their cooperation. This study was supported by the URF-QAU (2013-14), Pakistan.

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LEGENDS TO FIGURES:

FIG. 1. A. Pedigree of the family showing CLSS in fifth generation. The horizontal bar above the

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symbols denotes individuals who underwent a physical examination and an asterisk indicates the individuals who were available for molecular analyses. B. Identified

homozygous LRP4 mutation. (1). Sanger sequencing around the mutation in index

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subject; (2) in heterozygous sib.

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FIG. 2. Phenotypes of the affected individuals 505 (A-F), 504 (G-H), and 502 (I-K). C. Hypoplasia and posteriorly dislocated fibulae; D. Frontal bossing and bent ear; E. Hypoplastic shoulder and discrepancy in the length of humurii; G. Prominent forehead

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and clevicals; I. Surgically corrected cleft-lip/maxilla with mal-aligned teeth.

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Table 1: Phenotypes of the Affected Individuals 502 M, 12

F, 9

F, 6

Upper limbs Unrecognizable fingers Acromesomelic forearms Carpals/metacarpals synostosis Radio-ulnar sysnostosis Hypoplastic elbow joint Prominent clavicle and hypoplastic shoulder joint

+ + + + + +

+ + NA NA NA +

+ + + + + +

Lower limbs Short feet

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Difficulty in walking

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Thoracic scoliosis

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Facial and other features Broad forehead/frontal bossing High hair-line Deformed pinna Antevert/short nose Depressed nasal bridge Small pit/depression on nasal tip Cleft-lip Hypoplastic frontal teeth

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Ultrasonographic measurements (cm) Right kidney length Right kidney width Left kidney length Left kidney width

505

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+ 3 toes in right, 4 toes in left + NA

+



+

+ + +, left + + − + +

+ + + − + − −

+ + +, right + + − − −

+

NA

+

7.9 3.1 6.5 3.3

7.3 3.0 NV NV

8.7 3.3 NV NV

3 toes, bilaterally +

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Oligodactylous feet Synostosis of tarsals/metatarsals Hypoplastic knee joints

504

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Variables Gender, age

− −



3 toes bilaterally + +

+= feature present; – = feature absent; NA = not ascertained; NV = not visualized.

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Table 2: Anthropometric measurements of subjects 401 M, 35 UA 177.0 (+1 SD)

402 F, 33 UA 161.5

501 M, 18 UA 146.5 (< -1 SD)

502 M, 12 A 112 (< -3 SD)

58.0

58.0

55.0 (50%)

51.5 (+5 SD)

Standing height (cm)*

504 F, 9 A 99 (< -5 SD)

505 F, 6 A 80 (< -5 SD)

52.0 (50%)

49.0 (+5 SD)

46.5 (+5 SD)

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OFC (cm)**

503 F, 10 UA 122.0 (< -1 SD)

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Variables Gender, age Affection status

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A=affected; UA=unaffected; SD=standard deviation

*Growth standards compared with Pakistani subjects: Aziz S, Noor-Ul-Ain W, Majeed R, Khan MA, Qayum I, Ahmed I, Hosain K. Growth centile charts (anthropometric measurement) of Pakistani pediatric population. J. Pak. Med. Assoc. 62(4):367-77, 2012.

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**WHO child growth standard compared

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Supp. Table 1: Computational prediction of the variant

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Supplementary data:

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