Molecular Characterization of TPO gene mutations in Sudanese patients with Congenital Hypothyroidism

Abstract

Introduction

Congenital hypothyroidism is the most common congenital endocrine disorder

worldwide

.

Approximately 80

to 85% of cases are caused by defects in thyroid development

(dysgenesis),

the remaining 15 to

20% are due to errors of thyroid hormone biosynthesis

(dyshormonogenesis)

.

Congenital hypothyroidism is also the most common preven

table

cause of mental retardation. Its neurological defects can only be reversible if diagnosed and

treated

early. I

t

s incidence rate is

1

out of 3000/4000 live births worldwide However, t

his

incidence rate

is higher in

developing countries

such as Sudan

(

1 of 1400/2200 newborn

infants

)

in which its population is character

ized by consanguinity (25

-

70%).

The present study aimed to

assess the demographic and clinical pattern of congenital

hypothyroidism (dyshormonogenesis) in Sudan, to characterize

TPO

gene mutations in

Sudanese patients with Congenital Hypothyroidism (dyshormonogenesis

)

.

Methods

A total of 5

6

patients referred to Gaffar Ibn Auf Children Hospital presented with clinical

features suggesting congenital hypothyroidism (dyshormonogenesis) were enrolled in this

study. Demographic and clinical data was obtained by a predesigned questionnare.

Five ml

of venous blood were collected

in EDTA

tube

from each patient under complete

aseptic conditions for DNA extraction. All patients signed a written consent that has been

approved by Al Neelain University Ethical Committee prior to the study.

Genomic DNA was

extracted using Guanidine Chloride method

.

Detection of

TPO

gene was

performed by polymerase chain reaction (PCR) using

primers for selected exons 7, 8, 9, and

14.

Reaction products were electrophoresed on 1% agarose

–

TBE gels and visualized with

XIII

gel documentation system. Amplified region was directly sequenced to identify and

characterize mutations in

TPO

gene.

Data were analyzed using SPSS 13 software. Descriptive statistics (frequencies and

percentages) were obtained for categorical variables.

Genotype analysis was performed using

Bio Edit program,

Haploview program and Phyre bioscientific website for prediction of

protein structure and configuration.

Results

Most patients enrolled in this study (85.7%) a

re descendants of consanguineous

marriage

s.

There were 11

reported families,

comprising 74.3% of cases, with more than one affected

member

, o

f th

ose family

members,

68.6 % were in fact siblings. Patients from

consanguineous marriage had a 96.6% positive family history.

The majority of patients

(9

7.1%) developed complications.

According to the tribal origin,

the vast majority

of

patients

(65.7%) were from Afro

-

Asiatic tribes, whereas 34.3% were from Nilo

-

Saharan tribes.

There

was a large variation in the geographical distribution of patients. Biochemical analysis and

ultrasound findings were concordant with the genetic analysis results of patients.

Sequencing analysis

revealed apparent 18 single base changes

(missense m

utations)

in exons

7, 8, and 9, and a deletion at exon14.

Bioinformatics analysis showed strong L

inkage

D

isequilibrium

between two reported SNPs.

Therefore they can be considered as

potential

genetic

markers for disease association

studies

, population stud

ies and neonatal screening.

Conclusion

Consanguinity, tribal marriages, and positive family history are factors that increase the risk

of genetic disorders

.

Nineteen mutation including 8 novel mutations were detected. Strong

LD between two SNPs is a potent

ial g

enetic

marker for newborn screening of CH.